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Publication numberUS7429646 B1
Publication typeGrant
Application numberUS 09/533,262
Publication dateSep 30, 2008
Filing dateMar 22, 2000
Priority dateJun 5, 1995
Fee statusPaid
Also published asUS7824675, US20090092608
Publication number09533262, 533262, US 7429646 B1, US 7429646B1, US-B1-7429646, US7429646 B1, US7429646B1
InventorsJian Ni, Craig A. Rosen, Reiner L. Gentz
Original AssigneeHuman Genome Sciences, Inc.
Export CitationBiBTeX, EndNote, RefMan
External Links: USPTO, USPTO Assignment, Espacenet
Monoclonal, polyclonal, chimeric, humanize, fv, and or fab antibodies; hybridomas; vaccines; genetic engineering; isolated nucleic acids encoding a human TR2 receptor and two splice variants; medical diagnosis
US 7429646 B1
Abstract
The present invention relates to novel members of the Tumor Necrosis Factor family of receptors. The invention provides isolated nucleic acid molecules encoding a human TR2 receptor and two splice variants thereof. TR2 polypeptides are also provided as are vectors, host cells and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists and antagonists of TR2 receptor activity. Also provided are diagnostic methods for detecting disease states related to the aberrant states related to aberrant proliferation and differentiation of cells which express the TR2 receptors.
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Claims(41)
1. An isolated antibody which specifically binds to a polypeptide consisting of amino acids −38 to 245 of SEQ ID NO:26.
2. The isolated antibody of claim 1, wherein said antibody is a monoclonal antibody.
3. The isolated antibody of claim 1, wherein said antibody is a polyclonal antibody.
4. The isolated antibody of claim 1, wherein said antibody is a chimeric antibody.
5. The isolated antibody of claim 1, wherein said antibody is a humanized antibody.
6. The isolated antibody of claim 1, wherein said antibody is a single-chain Fv antibody.
7. The isolated antibody of claim 1, wherein said antibody is a Fab antibody.
8. A composition comprising the antibody of claim 1 and a pharmaceutically acceptable carrier.
9. An isolated antibody which binds to a polypeptide consisting of amino acids −38 to 162 of SEQ ID NO:26.
10. The isolated antibody of claim 9 which binds to a polypeptide consisting of amino acids 1 to 162 of SEQ ID NO:26.
11. A composition comprising the antibody of claim 9 and a pharmaceutically acceptable carrier.
12. The isolated antibody of claim 1, wherein said antibody is the product of an Fab expression library.
13. A method of producing the isolated antibody of claim 1, comprising:
(a) immunizing an animal with a polypeptide comprising amino acids −38 to 245 of SEQ ID NO:26; and
(b) recovering an antibody, which specifically binds, said polypeptide.
14. A hybridoma which produces the monoclonal antibody of claim 2.
15. A method of producing a monoclonal antibody which comprises:
(a) culturing the hybridoma of claim 14 under appropriate conditions; and
(b) isolating monoclonal antibody therefrom.
16. An isolated antibody which specifically binds to a polypeptide consisting of amino acids −38 to 162 of SEQ ID NO:26.
17. The isolated antibody of claim 16, wherein said antibody is a monoclonal antibody.
18. The isolated antibody of claim 16, wherein said antibody is a polyclonal antibody.
19. The isolated antibody of claim 16, wherein said antibody is a chimeric antibody.
20. The isolated antibody of claim 16, wherein said antibody is a humanized antibody.
21. The isolated antibody of claim 16, wherein said antibody is a single-chain Fv antibody.
22. The isolated antibody of claim 16, wherein said antibody is an Fab antibody fragment.
23. The isolated antibody of claim 16, wherein said antibody is a product of an Fab expression library.
24. A composition comprising the antibody of claim 16 and a pharmaceutically acceptable carrier.
25. A method of producing the isolated antibody of claim 16, comprising:
(a) immunizing an animal with a polypeptide comprising amino acids −38 to 162 of SEQ ID NO:26; and
(b) recovering an antibody which specifically binds said polypeptide.
26. A hybridoma which produces the monoclonal antibody of claim 17.
27. A method of producing a monoclonal antibody which comprises:
(a) culturing the hybridoma of claim 26 under appropriate conditions; and
(b) isolating monoclonal antibody therefrom.
28. An isolated antibody which specifically binds to a polypeptide consisting of amino acids 1 to 162 of SEQ ID NO:26.
29. The isolated antibody of claim 28, wherein said antibody is a monoclonal antibody.
30. The isolated antibody of claim 28, wherein said antibody is a polyclonal antibody.
31. The isolated antibody of claim 28, wherein said antibody is a chimeric antibody.
32. The isolated antibody of claim 28, wherein said antibody is a humanized antibody.
33. The isolated antibody of claim 28, wherein said antibody is a single-chain Fv antibody.
34. The isolated antibody of claim 28, wherein said antibody is an Fab antibody fragment.
35. The isolated antibody of claim 28, wherein said antibody is the product of an Fab expression library.
36. A composition comprising the antibody of claim 28 and a pharmaceutically acceptable carrier.
37. A method of producing the isolated antibody of claim 28, comprising:
(a) immunizing an animal with a polypeptide comprising amino acids 1 to 162 of SEQ ID NO:26; and
(b) recovering an antibody, which specifically binds, said polypeptide.
38. A hybridoma which produces the monoclonal antibody of claim 29.
39. A method of producing a monoclonal antibody which comprises:
(a) culturing the hybridoma of claim 38 under appropriate conditions; and
(b) isolating monoclonal antibody therefrom.
40. An isolated antibody which binds to a polypeptide having the sequence of SEQ ID NO:26.
41. An isolated antibody that immunospecifically binds a tumor necrosis factor receptor, wherein said receptor comprises a polypeptide having the sequence of SEQ ID NO:26.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims benefit to the filing dates of U.S. Provisional Application No. 60/147,383, filed Aug. 6, 1999; U.S. Provisional Application No. 60/135,169, filed May 20, 1999; U.S. Provisional Application No. 60/126,522, filed Mar. 26, 1999; and U.S. Provisional Application No. 60/125,683, filed Mar. 22, 1999, and is a continuation-in-part of U.S. application Ser. No. 08/741,095, filed Oct. 30, 1996, each of which is incorporated herein by reference; said 08/741,095 is a continuation-in-part of U.S. application Ser. No. 08/464,595, U.S. application Ser. No. 08/462,962, and U.S. application Ser. No. 08/462,315, each of which was filed Jun. 5, 1995 now abandoned, each of which is incorporated herein by reference; said U.S. application Ser. Nos. 08/464,595, 08/462,962 and 08/462,315 are each continuations-in-part of PCT/US95/05058, filed Apr. 27, 1995, which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel members of the Tumor Necrosis Factor (TNF) receptor family. More specifically, isolated nucleic acid molecules are provided encoding a human TNF receptor-related protein, referred to herein as the TR2 receptor of FIGS. 1A-1B, having considerable homology to murine CD40. Two different TR2 splice variants, referred to as TR2-SV1 and TR2-SV2, are also provided. TR2 polypeptides are also provided with homology to human type 2 TNF receptor (TNF-RII). Further provided are vectors, host cells and recombinant methods for producing the same. The invention also relates to both the inhibition and enhancement of functional activities of TR2 receptor polypeptides and diagnostic methods for detecting TR2 receptor gene expression.

2. Related Art

Human tumor necrosis factors α (TNF-α) and β (TNF-β or lymphotoxin) are related members of a broad class of polypeptide mediators, which includes the interferons, interleukins and growth factors, collectively called cytokines (Beutler, B. and Cerami, A., Annu. Rev. Immunol. 7:625-655 (1989)).

Tumor necrosis factor (TNF-α and TNF-β) was originally discovered as a result of its anti-tumor activity, however, now it is recognized as a pleiotropic cytokine playing important roles in a host of biological processes and pathologies. To date, there are ten known members of the TNF-related cytokine family, TNF-α, TNF-β (lymphotoxin-α), LT-β, TRAIL and ligands for the Fas receptor, CD30, CD27, CD40, OX40 and 4-1BB receptors. These proteins have conserved C-terminal sequences and variable N-terminal sequences which are often used as membrane anchors, with the exception of TNF-β. Both TNF-α and TNF-β function as homotrimers when they bind to TNF receptors.

TNF is produced by a number of cell types, including monocytes, fibroblasts, T-cells, natural killer (NK) cells and predominately by activated macrophages. TNF-α has been reported to have a role in the rapid necrosis of tumors, immunostimulation, autoimmune disease, graft rejection, producing an anti-viral response, septic shock, cerebral malaria, cytotoxicity, protection against deleterious effects of ionizing radiation produced during a course of chemotherapy, such as denaturation of enzymes, lipid peroxidation and DNA damage (Nata et al., J. Immunol. 136(7):2483 (1987)), growth regulation, vascular endothelium effects and metabolic effects. TNF-α also triggers endothelial cells to secrete various factors, including PAI-1, IL-1, GM-CSF and IL-6 to promote cell proliferation. In addition, TNF-α up-regulates various cell adhesion molecules such as E-Selectin, ICAM-1 and VCAM-1. TNF-α and the Fas ligand have also been shown to induce programmed cell death.

TNF-β has many activities, including induction of an antiviral state and tumor necrosis, activation of polymorphonuclear leukocytes, induction of class I major histocompatibility complex antigens on endothelial cells, induction of adhesion molecules on endothelium and growth hormone stimulation (Ruddle, N. and Homer, R., Prog Allergy 40:162-182 (1988)).

Both TNF-α and TNF-β are involved in growth regulation and interact with hemopoietic cells at several stages of differentiation, inhibiting proliferation of various types of precursor cells, and inducing proliferation of immature myelomonocytic cells. Porter, A., Tibtech 9:158-162 (1991).

Recent studies with “knockout” mice have shown that mice deficient in TNF-β production show abnormal development of the peripheral lymphoid organs and morphological changes in spleen architecture (reviewed in Aggarwal et al, Eur Cytokine Netw, 7(2):93-124 (1996)). With respect to the lymphoid organs, the popliteal, inguinal, para-aortic, mesenteric, axillary and cervical lymph nodes failed to develop in TNF-β −/− mice. In addition, peripheral blood from TNF-β −/− mice contained a three fold reduction in white blood cells as compared to normal mice. Peripheral blood from TNF-β −/− mice, however, contained four fold more B cells as compared to their normal counterparts. Further, TNF-β, in contrast to TNF-α has been shown to induce proliferation of EBV-infected B cells. These results indicate that TNF-β is involved in lymphocyte development.

The first step in the induction of the various cellular responses mediated by TNF-α or TNF-β is their binding to specific cell surface or soluble receptors. Two distinct TNF receptors of approximately 55-KDa (TNF-R1) and 75-KDa (TNF-RII) have been identified (Hohman et al., J Biol. Chem., 264:14927-14934 (1989)), and human and mouse cDNAs corresponding to both receptor types have been isolated and characterized (Loetscher et al., Cell, 61:351 (1990)). Both TNF-Rs share the typical structure of cell surface receptors including extracellular, transmembrane and intracellular regions.

These molecules exist not only in cell bound forms, but also in soluble forms, consisting of the cleaved extra-cellular domains of the intact receptors (Nophar et al, EMBO Journal, 9(10):3269-76 (1990)) and otherwise intact receptors wherein the transmembrane domain is lacking. The extracellular domains of TNF-RI and TNF-RII share 28% identity and are characterized by four repeated cysteine-rich motifs with significant intersubunit sequence homology. The majority of cell types and tissues appear to express both TNF receptors and both receptors are active in signal transduction, however, they are able to mediate distinct cellular responses. Further, TNF-RII was shown to exclusively mediate human T-cell proliferation by TNF as shown in PCT WO 94/09137.

TNF-RI dependent responses include accumulation of C-FOS, IL-6, and manganese superoxide dismutase mRNA, prostaglandin E2 synthesis, IL-2 receptor and MHC class I and II cell surface antigen expression, growth inhibition, and cytotoxicity. TNF-RI also triggers second messenger systems such as phospholipase A2, protein kinase C, phosphatidylcholine-specific phospholipase C and sphingomyelinase (Pfefferk et al., Cell, 73:457-467 (1993)).

Several interferons and other agents have been shown to regulate the expression of TNF receptors. Retinoic acid, for example, has been shown to induce the production of TNF receptors in some cells type while down regulating production in other cells. In addition, TNF-α has been shown to affect the localization of both types of receptor. TNF-α induces internalization of TNF-RI and secretion of TNF-RII (reviewed in Aggarwal et al., supra). Thus, the production and localization of both TNF-Rs are regulated by a variety of agents.

Both the yeast two hybrid system and co-precipitation and purification have been used to identify ligands which associate with both types of the TNF-Rs (reviewed in Aggarwal et al., supra and Vandenabeele et al., Trends in Cell Biol. 5:392-399 (1995)). Several proteins have been identified which interact with the cytoplasmic domain of a murine TNF-R. Two of these proteins appear to be related to the baculovirus inhibitor of apoptosis, suggesting a direct role for TNF-R in the regulation of programmed cell death.

SUMMARY OF THE INVENTION

The present invention provides isolated nucleic acid molecules comprising, or alternatively consisting of, polynucleotides encoding TR2 receptors and splice variants thereof having the amino acid sequences shown in SEQ ID NO:26, FIGS. 1A-1B (SEQ ID NO:2), FIGS. 4A-4B (SEQ ID NO:5) and FIGS. 7A-7B (SEQ ID NO:8) or the amino acid sequence encoded by the cDNA encoding the TR2 receptors deposited as ATCC Deposit Numbers 97059, 97058 and 97057 on Feb. 13, 1995. The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of TR2 polypeptides or peptides by recombinant techniques.

The invention further provides isolated TR2 polypeptides having amino acid sequences encoded by the polynucleotides described herein.

The present invention also provides a screening method for identifying compounds capable of enhancing or inhibiting a cellular response induced by TR2 receptors, which involves contacting cells which express TR2 receptors with the candidate compound, assaying a cellular response, and comparing the cellular response to a standard cellular response, the standard being assayed when contact is made in absence of the candidate compound; whereby, an increased cellular response over the standard indicates that the compound is an agonist and a decreased cellular response over the standard indicates that the compound is an antagonist.

In another aspect, a screening assay for agonists and antagonists is provided which involves determining the effect a candidate compound has on the binding of cellular ligands to TR2 receptors. In particular, the method involves contacting TR2 receptors with a ligand polypeptide and a candidate compound and determining whether ligand binding to the TR2 receptors is increased or decreased due to the presence of the candidate compound.

The invention further provides a diagnostic method useful during diagnosis or prognosis of a disease states resulting from aberrant cell proliferation due to alterations in TR2 receptor expression.

An additional aspect of the invention is related to a method for treating an individual in need of an increased level of a TR2 receptor activity in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of isolated TR2 polypeptides of the invention or an agonist thereof.

A still further aspect of the invention is related to a method for treating an individual in need of a decreased level of a TR2 receptor activity in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of a TR2 receptor antagonist.

The invention additionally provides soluble forms of the polypeptides of the present invention. Soluble peptides are defined by amino acid sequences wherein the sequence comprises, or alternatively consists of, the polypeptide sequences lacking a transmembrane domain. Such soluble forms of the TR2 receptors are useful as antagonists of the membrane bound forms of the receptors.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-1B shows the nucleotide (SEQ ID NO:1) and deduced amino acid (SEQ ID NO:2) sequences of a TR2 receptor. The protein has a predicted leader sequence of about 36 amino acid residues (underlined) (amino acid residues −36 to −1 in SEQ ID NO:2) and a deduced molecular weight of about 30,417 kDa. It is further predicted that amino acid residues from about 37 to about 200 (amino acid residues 1 to 164 in SEQ ID NO:2) constitute the extracellular domain; from about 201 to about 225 (amino acid residues 165 to 189 in SEQ ID NO:2) the transmembrane domain (underlined); and from about 226 to about 283 (amino acid residues 190 to 247 in SEQ ID NO:2) the intracellular domain. Two potential asparagine-linked glycosylation sites are located at amino acid positions 110 and 173 (amino acid residues 74 to 137 in SEQ ID NO:2).

FIG. 2 shows the regions of similarity between the amino acid sequences of the TR2 receptor protein of FIG. 1A-1B and a murine CD40 protein (SEQ ID NO:3).

FIG. 3 shows an analysis of the TR2 receptor amino acid sequence of FIG. 1A-1B. Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown. In the “Antigenic Index—Jameson-Wolf” graph, amino acid residues 39 to 70, 106 to 120, 142 to 189 and 276 to 283 in FIG. 1A-1B (amino acid residues 3 to 34, 70 to 84, 106 to 153 and 240 to 247 in SEQ ID NO:2) correspond to the shown highly antigenic regions of the TR2 receptor protein.

FIG. 4A-4B shows the nucleotide (SEQ ID NO:4) and deduced amino acid (SEQ ID NO:5) sequences of the TR2-SV1 receptor. The protein has a predicted leader sequence of about 36 amino acid residues (underlined) (amino acid residues −36 to −1 in SEQ ID NO:5) and a deduced molecular weight of about 19.5 kDa.

FIG. 5 shows the regions of similarity between the amino acid sequences of the full-length TR2-SV1 receptor protein and a human type 2 TNF receptor (SEQ ID NO:6).

FIG. 6 shows an analysis of the TR2-SV1 receptor amino acid sequence. Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown. In the “Antigenic Index—Jameson-Wolf” graph, amino acid residues 39 to 70, 99 to 136 and 171 to 185 in FIG. 4A-4B (amino acid residues 3 to 34, 63 to 100 and 135 to 149 in SEQ ID NO:5) correspond to the shown highly antigenic regions of the TR2-SV1 receptor protein.

FIG. 7A-7B shows the nucleotide (SEQ ID NO:7) and deduced amino acid (SEQ ID NO:8) sequences of the TR2-SV2 receptor. This protein lacks a putative leader sequence and has a deduced molecular weight of about 14 kDa.

FIG. 8 shows the regions of similarity between the amino acid sequences of the TR2-SV2 receptor protein and a human type 2 TNF receptor (SEQ ID NO:9).

FIG. 9 shows an analysis of the TR2-SV2 receptor amino acid sequence. Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown. In the “Antigenic Index—Jameson-Wolf” graph, amino acid residues 56 to 68 and 93 to 136 in FIG. 7A-7B (SEQ ID NO:8) correspond to the shown highly antigenic regions of the TR2-SV2 receptor protein.

FIG. 10 shows the regions of similarity between the amino acid sequences of the TR2 receptor protein of FIG. 1A-1B and the TR2-SV1 receptor protein of FIG. 4A-4B.

FIG. 11 shows the regions of similarity between the amino acid sequences of the TR2 receptor protein of FIG. 1A-1B and the TR2-SV2 receptor protein of FIG. 7A-7B.

FIG. 12 shows the regions of similarity between the amino acid sequences of the TR2-SV1 and the TR2-SV2 receptor proteins.

FIG. 13A-13C shows the regions of similarity between the nucleotide sequences encoding the TR2 receptor protein of FIG. 1A-1B and the TR2-SV1 receptor protein of FIG. 4A-4B.

FIG. 14A-14C shows the regions of similarity between the nucleotide sequences encoding the TR2 receptor protein of FIG. 1A-1B and the TR2-SV2 receptor protein of FIG. 7A-7B.

FIG. 15A-15E shows the regions of similarity between the nucleotide sequences encoding the TR2-SV1 and the TR2-SV2 receptor proteins.

FIG. 16 shows an alignment of the amino acid sequence of the TR2 receptor of FIG. 1A-1B (SEQ ID NO:2) with other TNFR family members. The amino acid sequence of TR2 was aligned with those of TNFR-I (SEQ ID NO:10), TNFR-II (SEQ ID NO:11), CD40 (SEQ ID NO:12) and 4-1BB (SEQ ID NO:13) on the basis of sequence homology and conserved cysteine residues. Cysteine repeat regions are defined by amino acid residues 5 to 40, 41 to 84, 85 to 127, and 128 to 166 in SEQ ID NO:2, respectively referred to as cysteine repeat regions A-D.

FIG. 17 shows the effect of TR2 on B cell in vitro proliferation. B lymphocytes were purified from human tonsils by immunomagnetic selection. Cells were cultured for 72 hours followed by a 24 hour 3H thymidine pulse in RPMI 1640 medium added with 10% FBS, 4 mM 1-glutamine, 5×10−5 M 2ME, 100 U/ml Penicillin, 100 μg/ml Streptomycin, and the indicated factors.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides isolated nucleic acid molecules comprising, or alternatively consisting of, polynucleotides encoding a TR2 polypeptide (FIG. 1A-1B (SEQ ID NO:2)) and splice variants thereof, TR2-SV1 (FIG. 4A-4B (SEQ ID NO:5)) and TR2-SV2 (FIG. 7A-7B (SEQ ID NO:8)), the amino acid sequences of which were determined by sequencing cDNAs. The TR2 protein shown in FIG. 1A-1B shares sequence homology with the murine CD40 receptor (FIG. 2 (SEQ ID NO:3)). On Feb. 13, 1995 a deposit was made at the American Type Culture Collection, 10801 University Blvd., Manassas, Va. 20110-2209, USA, and given ATCC Accession No. 97059. The nucleotide sequence shown in FIG. 1A-1B (SEQ ID NO:1) was obtained by sequencing a cDNA which is believed to contain the same amino acid coding sequences as the cDNA contained in the deposited plasmid assigned ATCC Accession No. 97059 (Clone ID HLHAB49).

The TR2 receptors of the present invention include several allelic variants containing alterations in at least four nucleotides and two amino acids. Nucleotide sequence variants which have been identified include either guanine or adenine at nucleotide 314 and either thymine or cytosine at nucleotides 386, 624 and 627 shown in FIG. 1A-1B (SEQ ID NO:1). While the identified alteration at nucleotides 624 and 627 are silent, the alteration at nucleotide 386 results in the codon at nucleotides 385 to 387 encoding either serine or phenylalanine and the alteration at nucleotide 314 results in the codon at nucleotides 313 to 315 encoding either lysine or arginine.

The nucleotide sequences shown in FIG. 4A-4B (SEQ ID NO:4) and FIG. 7A-7B (SEQ ID NO:7) were also obtained by sequencing cDNAs deposited on Feb. 13, 1995 at the American Type Culture Collection and given accession numbers 97058 (TR2-SV1) and 97057 (TR2-SV2), respectively. The deposited cDNAs are contained in the pBluescript SK(−) plasmid (Stratagene, LaJolla, Calif.).

As used herein the phrase “splice variant” refers to cDNA molecules produced from a RNA molecules initially transcribed from the same genomic DNA sequence which have undergone alternative RNA splicing. Alternative RNA splicing occurs when a primary RNA transcript undergoes splicing, generally for the removal of introns, which results in the production of more than one mRNA molecule each of which may encode different amino acid sequences. The term “splice variant” also refers to the proteins encoded by the above cDNA molecules.

As used herein, “TR2 proteins”, “TR2 receptors”, “TR2 receptor proteins” and “TR2 polypeptides” refer to all proteins resulting from the alternate splicing of the genomic DNA sequences encoding proteins having regions of amino acid sequence identity and receptor activity which correspond to the proteins shown in SEQ ID NO:26, FIG. 1A-1B (SEQ ID NO:2), FIG. 4A-4B (SEQ ID NO:5) or FIG. 7A-7B (SEQ ID NO:8), as well as TR2 allellic variants. The TR2 proteins shown in SEQ ID NO:26 and FIG. 1A-1B, the TR2-SV1 protein shown FIG. 4A-4B, and the TR2-SV2 protein shown in FIG. 7A-7B are examples of such receptor proteins.

Nucleic Acid Molecules

Unless otherwise indicated, all nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer (such as the Model 373 from Applied Biosystems, Inc.), and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined as above. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art. As is also known in the art, a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion.

Using the information provided herein, such as the nucleotide sequence in SEQ ID NO:26, FIG. 1A-1B, FIG. 4A-4B or FIG. 7A-7B, nucleic acid molecules of the present invention encoding TR2 polypeptides may be obtained using standard cloning and screening procedures, such as those used for cloning cDNAs using mRNA as starting material. Illustrative of the invention, the nucleic acid molecule described in FIG. 1A-1B (SEQ ID NO:1) was discovered in a cDNA library derived from activated human T-lymphocytes. The nucleic acid molecules described in FIG. 4A-4B (SEQ ID NO:4) and FIG. 7A-7B (SEQ ID NO:7) were discovered in cDNAs library derived from human fetal heart and human stimulated monocytes, respectively.

As described in Example 6, TR2 mRNA was detected in numerous tissues including lung, spleen and thymus and may be ubiquitously expressed in human cells. TR2RNA was also found to be expressed in B lymphocytes (CD 19+), both CD4+ (TH1 and TH2 clones) and CD8+ T lymphocytes, monocytes and endothelial cells.

As also noted in Example 6, the production of TR2 mRNA was inducible in MG 63 cells by TNFα. Further, the accumulation of TR2 mRNA was observed in HL60, U937 and THP1 cells upon PMA or DMSO treatment. PMA and DMSO are agents known to induce differentiation of these three cell types.

The determined nucleotide sequence of the TR2 cDNA of FIG. 1A-1B (SEQ ID NO:1) contains an open reading frame encoding a protein of about 283 amino acid residues, with a predicted leader sequence of about 36 amino acid residues, and a deduced molecular weight of about 30,417 kDa. The amino acid sequence of the predicted mature TR2 receptor is shown in FIG. 1A-1B from amino acid residue about 37 to residue about 283 (amino acid residues 1 to 247 in SEQ ID NO:2). In this context “about” includes the particularly recited value and values larger or smaller by several (5, 4, 3, 2, or 1) amino acids. As noted in Example 6, the location of the leader sequence cleavage site was confirmed for a TR2-Fc fusion protein and found to be between amino acids 36 and 37 shown in FIG. 1A-1B (amino acid residues −1 to 1 in SEQ ID NO:2). The TR2 protein shown in FIG. 1A-1B (SEQ ID NO:2) is about 29% identical and about 47% similar to the murine CD40 protein shown in SEQ ID NO:3 (see FIG. 2).

Similarly, the determined cDNA nucleotide sequences of the TR2-SV1 splice variant of TR2 (FIG. 4A-4B (SEQ ID NO:4)) contains an open reading frame encoding a protein of about 185 amino acid residues, with a predicted leader sequence of about 36 amino acid residues, and a deduced molecular weight of about 19.5 kDa. The amino acid sequence of the predicted mature TR2-SV1 receptor is shown in FIG. 4A-4B (SEQ ID NO:5) from amino acid residue about 37 to residue about 185 (amino acid residues 1 to 149 in (SEQ ID NO:5). In this context “about” includes the particularly recited value and values larger or smaller by several (5, 4, 3, 2, or 1) amino acids. The TR2-SV1 protein shown in FIG. 4A-4B (SEQ ID NO:5) is about 25% identical and about 48% similar to the human type 2 TNF receptor protein shown in SEQ ID NO:6 (see FIG. 5).

The determined cDNA nucleotide sequences of the TR2-SV2 splice variant of TR2 (FIG. 7A-7B (SEQ ID NO:7)) contains an open reading frame encoding a protein of about 136 amino acid residues, without a predicted leader sequence, and a deduced molecular weight of about 14 kDa. The amino acid sequence of the predicted TR2-SV2 receptor is shown in FIG. 7A-7B (SEQ ID NO:8) from amino acid residue about 1 to residue about 136. In this context “about” includes the particularly recited value and values larger or smaller by several (5, 4, 3, 2, or 1) amino acids. The TR2-SV2 protein shown in FIG. 7A-7B (SEQ ID NO:8) is about 27% identical and about 45% similar to the human type 2 TNF receptor protein shown in SEQ ID NO:9 (see FIG. 8).

A comparison of both the nucleotide and amino acid sequences of the TR2, TR2-SV1 and TR2-SV2 receptor proteins shown in FIG. 1A-1B, FIG. 4A-4B and FIG. 7A-7B shows several regions of near identity. While the amino acid sequence of the TR2 receptor protein, shown in FIG. 1A-1B (SEQ ID NO:2), is about 60% identical and about 73% similar to the amino acid sequence of the TR2-SV1 receptor protein, shown in FIG. 4A-4B (SEQ ID NO:5), in approximately the first one hundred amino acids of their respective sequences the two proteins differ in one location (FIG. 10).

Similarly, the amino acid sequence of the TR2 receptor protein of FIG. 1A-1B (SEQ ID NO:2) is about 60% identical and about 71% similar to the amino acid sequence of the TR2-SV2 receptor protein, shown in FIG. 7A-7B (SEQ ID NO:8); however, the two proteins are almost identical over a 60 amino acid stretch in the central portion of the TR2-SV2 protein (FIG. 11).

In contrast, the TR2-SV1 and TR2-SV2 proteins are only about 20% identical and about 38% similar at the amino acid level to each other. Unlike the comparisons of either of these proteins to the TR2 protein shown in FIG. 1A-1B (SEQ ID NO:2), these proteins share their homology over the entire 136 amino acid sequence of the TR2-SV2 protein (FIG. 12).

With respect to their nucleotide sequences of the cDNAs encoding the disclosed TR2 proteins, a comparison of these sequences indicates that the TR2 cDNAs share large regions of near identity at the nucleic acid level (FIG. 13A-13C, FIG. 14A-14C and FIG. 15A-15E). The cDNA sequences encoding the TR2 and TR2-SV1 proteins, for example, share large regions of near identity in their nucleotide sequences which encode both the N termini of the respective proteins and their 5′ and 3′ noncoding regions (FIG. 13A-13C). Further, the nucleotide sequences of the cDNAs encoding the TR2-SV1 and TR2-SV2 proteins share considerable homology but this identity is limited to their 3′ regions well beyond their respective coding sequences (FIG. 15A-15E).

Such regions of near identity between two different cDNA sequences, when maintained over an extended stretch of sequence, indicates to one skilled in the art that the respective molecules were originally transcribed from the same genomic DNA sequence. One skilled in the art would further recognize that, since more than one codon can encode the same amino acid, identity between two proteins at the amino acid level does not necessarily mean that the DNA sequences encoding those proteins will share similar regions of identity. The above data indicates that the TR2 receptors of the present invention are transcribed from a single genomic DNA sequence and represent multiple splice variants of one initial RNA transcript.

Related proteins which are produced from alternately spliced RNA, referred to as splice variants, are known in the art. The transcript of the src gene, for example, undergoes alternate RNA splicing to produce cell type specific products. In most cells the Src protein consists of 533 amino acids while in nerve cells an additional short exon is included in the mRNA resulting in a protein of 539 amino acids. See Alberts, B. e/al., MOLECULAR BIOLOGY OF THE CELL (3rd Edition, Garland Publishing, Inc., 1994), 455. Similarly, sex specific mRNA transcripts have been identified in Drosophila where alternate mRNA splicing results in a protein named Dsx which is approximately 550 amino acids in length in males and 430 amino acids in length in females. These two splice variant proteins share a common core sequence of about 400 amino acids. See id. at 457.

In the present instance, the TR2 receptor protein shown in FIG. 1A-1B (SEQ ID NO:2) is believed to be the full-length polypeptide encoded by the RNA from which the TR2 receptor proteins are translated. The RNA encoding the TR2-SV1 splice variant shown in FIG. 4A-4B (SEQ ID NO:5) is believed to contain an insertion in the region encoding amino acid residue 102 of the amino acid sequence shown in FIG. 1A-1B and a deletion in the region encoding amino acid residue 184 of the amino acid sequence shown in FIG. 1A-1B. The RNA encoding the TR2-SV2 splice variant shown in FIG. 7A-7B is believed to begin with the nucleotide sequence encoding amino acid residue 102 of the amino acid sequence shown in FIG. 1A-1B and contain insertions in the regions encoding amino acid residues 184 and 243 of the amino acid sequence shown in FIG. 1A-1B.

As indicated, the present invention also provides the mature forms of the TR2 receptors of the present invention. According to the signal hypothesis, proteins secreted by mammalian cells have a signal or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Most mammalian cells and even insect cells cleave secreted proteins with the same specificity. However, in some cases, cleavage of a secreted protein is not entirely uniform, which results in two or more mature species on the protein. Further, it has long been known that the cleavage specificity of a secreted protein is ultimately determined by the primary structure of the complete protein, that is, it is inherent in the amino acid sequence of the polypeptide. Therefore, the present invention provides nucleotide sequences encoding mature TR2 polypeptides having the amino acid sequences encoded by the cDNAs contained in the deposits identified as ATCC Deposit Numbers 97059 and 97058 and as shown in SEQ ID NO:26, FIG. 1A-1B (SEQ ID NO:2) and FIG. 4A-4B (SEQ ID NO:5). By the mature TR2 polypeptides having the amino acid sequences encoded by the cDNAs contained in the deposits identified as ATCC Deposit Numbers 97059 and 97058 is meant the mature form(s) of the TR2 receptors produced by expression in a mammalian cell (e.g., COS cells, as described below) of the complete open reading frame encoded by the human DNA sequence of the cDNA contained in the deposited plasmids.

The invention also provides nucleic acid sequences encoding the TR2-SV2 receptor protein of FIG. 7A-7B (SEQ ID NO:8), having the amino acid sequence encoded by the cDNA contained in ATCC Deposit Number 97057, which does not contain a secretory leader sequence.

Methods for predicting whether a protein has a secretory leader as well as the cleavage point for that leader sequence are available. For instance, the methods of McGeoch (Virus Res. 3:271-286 (1985)) and von Heinje (Nucleic Acids Res. 14:4683-4690 (1986)) can be used. The accuracy of predicting the cleavage points of known mammalian secretory proteins for each of these methods is in the range of 75-80%. von Heinje, supra. However, the two methods do not always produce the same predicted cleavage point(s) for a given protein.

In the present case, the predicted amino acid sequences of the complete TR2 polypeptides shown in FIG. 1A-1B (SEQ ID NO:2), FIG. 4A-4B (SEQ ID NO:5) and FIG. 7A-7B (SEQ ID NO:8) were analyzed by a computer program (“PSORT”) (K. Nakai and M. Kanehisa, Genomics 14:897-911 (1992)), which is an expert system for predicting the cellular location of a protein based on the amino acid sequence. As part of this computational prediction of localization, the methods of McGeoch and von Heinje are incorporated. The analysis by the PSORT program predicted the cleavage sites between amino acids −1 and 1 in SEQ ID NO:2 and SEQ ID NO:5. Thereafter, the complete amino acid sequences were further analyzed by visual inspection, applying a simple form of the (−1,−3) rule of von Heine. von Heinje, supra. Thus, the leader sequences for the TR2 protein shown in SEQ ID NO:2 and the TR2-SV1 protein are predicted to consist of amino acid residues −36 to −1 in both SEQ ID NO:2 and SEQ ID NO:5, while the predicted mature TR2 proteins consist of amino acid residues 1 to 247 for the TR2 protein shown in SEQ ID NO:2 and residues 1 to 149 for the TR2-SV1 protein shown in SEQ ID NO:5.

As noted in Example 6, the cleavage site of the leader sequence of a TR2-Fc fusion protein was confirmed using amino acid analysis of the expressed fusion protein. This fusion protein was found to begin at amino acid 37, which corresponds to amino acid 1 in SEQ ID NO:2 and SEQ ID NO:5, indicating that the cleavage site of the leader sequence is between amino acids 36 and 37 in this protein (corresponding to amino acid residues −1 to 1 in SEQ ID NO:2 and SEQ ID NO:5).

As one of ordinary skill would appreciate, however, due to the possibilities of sequencing errors, as well as the variability of cleavage sites for leaders in different known proteins, the TR2 receptor polypeptide encoded by the cDNA of ATCC Deposit Number 97059 comprises about 283 amino acids, but may be anywhere in the range of 250 to 316 amino acids; and the leader sequence of this protein is about 36 amino acids, but may be anywhere in the range of about 30 to about 42 amino acids. Similarly, the TR2-SV1 receptor polypeptide encoded by the cDNA of ATCC Deposit Number 97058 comprises about 185 amino acids, but may be anywhere in the range of 163-207 amino acids; and the leader sequence of this protein is about 36 amino acids, but may be anywhere in the range of about 30 to about 42 amino acids. Further, the TR2-SV2 receptor polypeptide encoded by the cDNA of ATCC Deposit Number 97057 comprises about 136 amino acids, but may be anywhere in the range of 120-152 amino acids. In this context “about” includes the particularly recited value and values larger or smaller by several (5, 4, 3, 2, or 1) amino acids.

The leader sequences for the TR2 protein shown in SEQ ID NO:26 is predicted to consist of amino acid residues −38 to −1 in SEQ ID NO:26, while the predicted mature TR2 protein consists of amino acid residues 1 to 245 in SEQ ID NO:26.

As indicated, nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced synthetically. The DNA may be double-stranded or single-stranded. Single-stranded DNA or RNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand.

By “isolated” nucleic acid molecule(s) is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment. For example, recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.

However, a nucleic acid contained in a clone that is a member of a library (e.g., a genomic or cDNA library) that has not been isolated from other members of the library (e.g., in the form of a homogeneous solution containing the clone and other members of the library) or a chromosome isolated or removed from a cell or a cell lysate (e.g., a “chromosome spread,” as in a karyotype), is not “isolated” for the purposes of the invention. As discussed further herein, isolated nucleic acid molecules according to the present invention may be produced naturally, recombinantly, or synthetically.

Isolated nucleic acid molecules of the present invention include DNA molecules comprising, or alternatively consisting of, an open reading frame (ORF) shown in SEQ ID NO:26 or FIG. 1A-1B (SEQ ID NO:1); DNA molecules comprising, or alternatively consisting of, the coding sequence for the mature TR2 receptor shown in SEQ ID NO:26 (last 245 amino acids) or FIG. 1A-1B (SEQ ID NO:2) (last 247 amino acids); and DNA molecules which comprise, or alternatively consist of, a sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode the TR2 receptor protein shown in SEQ ID NO:26 or FIG. 1A-1B (SEQ ID NO:2). Of course, the genetic code is well known in the art. Thus, it would be routine for one skilled in the art to generate such degenerate variants.

Similarly, isolated nucleic acid molecules of the present invention include DNA molecules comprising, or alternatively consisting of, an open reading frame (ORF) shown in FIG. 4A-4B (SEQ ID NO:4); DNA molecules comprising, or alternatively consisting of, the coding sequence for the mature TR2-SV1 receptor shown in FIG. 4A-4B (SEQ ID NO:5) (last 149 amino acids); and DNA molecules which comprise, or alternatively consist of, a sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode the TR2-SV1 receptor.

Further, isolated nucleic acid molecules of the present invention include DNA molecules comprising, or alternatively consisting of, an open reading frame (ORF) shown in FIG. 7A-7B (SEQ ID NO:7) and DNA molecules which comprise, or alternatively consist of, a sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode the TR2-SV2 receptor.

In another aspect, the invention provides isolated nucleic acid molecules encoding the TR2, TR2-SV1 and TR2-SV2 polypeptides having the amino acid sequences encoded by the cDNAs contained in the plasmid deposited as ATCC Deposit No. 97059, 97058 and 97057, respectively, on Feb. 13, 1995. In a further embodiment, these nucleic acid molecules will encode a mature polypeptide or the full-length polypeptide lacking the N-terminal methionine. The invention further provides isolated nucleic acid molecules having the nucleotide sequences shown in SEQ ID NO:25, FIG. 1A-1B (SEQ ID NO:1), FIG. 4A-4B (SEQ ID NO:4), and FIG. 7A-7B (SEQ ID NO:7); the nucleotide sequences of the cDNAs contained in the above-described deposited cDNAs; or nucleic acid molecules having a sequence complementary to one of the above sequences. Such isolated molecules, particularly DNA molecules, are useful, for example, as probes for gene mapping, by in situ hybridization with chromosomes, and for detecting expression of the TR2 receptor genes of the present invention in human tissue, for instance, by Northern blot analysis.

Further embodiments of the invention include isolated nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide having a nucleotide sequence at least 80% identical, and more preferably at least 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to (a) a nucleotide sequence encoding the TR2 polypeptide having the complete amino acid sequence shown in SEQ ID NO:26, FIG. 1A-1B (amino acid residues −36 to 247 in SEQ ID NO:2), FIG. 4A-4B (amino acid residues −36 to 149 in SEQ ID NO:5), or FIG. 7A-7B (amino acid residues 1 to 136 in SEQ ID NO:8); (b) a nucleotide encoding the complete amino sequence shown in SEQ ID NO:26, FIG. 1A-1B (amino acid residues −35 to 247 in SEQ ID NO:2), FIG. 4A-4B (amino acid residues −35 to 149 in SEQ ID NO:5), or FIG. 7A-7B (amino acid residues 2 to 136 in SEQ ID NO:8) but lacking the N-terminal methionine; (c) a nucleotide sequence encoding the mature TR2 receptors (full-length polypeptide with any attending leader sequence removed) having the amino acid sequence at positions from about 1 to about 245 in SEQ ID NO:26, from about 37 to about 283 in FIG. 1A-1B (amino acid residues 1 to 247 in SEQ ID NO:2) or the amino acid sequence at positions from about 37 to about 185 in FIG. 4A-4B (amino acid residues 1 to 149 in SEQ ID NO:5), or the amino acid sequence at positions from about 1 to about 136 in FIG. 7A-7B (SEQ ID NO:8); (d) a nucleotide sequence encoding the TR2, TR2-SV1 or TR2-SV2 polypeptides having the complete amino acid sequence including the leader encoded by the cDNAs contained in ATCC Deposit Numbers 97059, 97058, and 97057, respectively; (e) a nucleotide sequence encoding the mature TR2 or TR2-SV1 receptors having the amino acid sequences encoded by the cDNAs contained in ATCC Deposit Numbers 97059 and 97058, respectively; (f) a nucleotide sequence encoding the TR2 or TR2-SV1 receptor extracellular domain; (g) a nucleotide sequence encoding the TR2 receptor transmembrane domain; (h) a nucleotide sequence encoding the TR2 receptor intracellular domain; (i) a nucleotide sequence encoding the TR2 receptor extracellular and intracellular domains with all or part of the transmembrane domain deleted; and (j) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), or (i). In this context “about” includes the particularly recited value and values larger or smaller by several (5, 4, 3, 2, or 1) amino acids.

By a polynucleotide having a nucleotide sequence at least, for example, 95% “identical” to a reference nucleotide sequence encoding a TR2 receptor polypeptide is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five mismatches per each 100 nucleotides of the reference nucleotide sequence encoding a TR2 receptor. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mismatches of the reference sequence may occur at the 5′ or 3′ terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. The reference (query) sequence may be the entire TR2 receptor encoding nucleotide sequence shown in SEQ ID NO:25, FIG. 1A-1B (SEQ ID NO:1), FIG. 4A-4B (SEQ ID NO:4), or FIG. 7A-7B (SEQ ID NO:7) or any TR2 receptor polynucleotide fragment (e.g., a polynucleotide encoding the amino acid sequence of any of the TR2 receptor N- and/or C-terminal deletions described herein), variant, derivative or analog, as described herein.

As a practical matter, whether any particular nucleic acid molecule is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the encoding nucleotide sequence shown in SEQ ID NO:25, FIG. 1A-1B (SEQ ID NO:1), FIG. 4A-4B (SEQ ID NO:4), or FIG. 7A-7B (SEQ ID NO:7), or to the nucleotide sequence of the deposited cDNAs, can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711). Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), to find the best segment of homology between two sequences. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.

In a specific embodiment, the identity between a reference (query) sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, is determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). Preferred parameters used in a FASTDB alignment of DNA sequences to calculate percent identity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30, Randomization Group Length=0, CutoffScore=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the length of the subject nucleotide sequence, whichever is shorter. According to this embodiment, if the subject sequence is shorter than the query sequence because of 5′ or 3′ deletions, not because of internal deletions, a manual correction is made to the results to take into consideration the fact that the FASTDB program does not account for 5′ and 3′ truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the 5′ or 3′ ends, relative to the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are 5′ and 3′ of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. A determination of whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This corrected score is what is used for the purposes of this embodiment. Only bases outside the 5′ and 3′ bases of the subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score. For example, a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity. The deletions occur at the 5′ end of the subject sequence and therefore, the FASTDB alignment does not show a matched/alignment of the first 10 bases at 5′ end. The 10 unpaired bases represent 10% of the sequence (number of bases at the 5′ and 3′ ends not matched/total number of bases in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%. In another example, a 90 base subject sequence is compared with a 100 base query sequence. This time the deletions are internal deletions so that there are no bases on the 5′ or 3′ of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only bases 5′ and 3′ of the subject sequence which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are made for the purposes of this embodiment.

The present application is directed to nucleic acid molecules at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequences disclosed herein, (e.g., encoding a polypeptide having the amino acid sequence of an N and/or C terminal deletion disclosed herein, such as, for example, a nucleic acid molecule encoding amino acids 50 to 283 of SEQ ID NO:2), irrespective of whether they encode a polypeptide having a TR2 receptor functional activity. This is because even where a particular nucleic acid molecule does not encode a polypeptide having TR2 receptor activity, one of skill in the art would still know how to use the nucleic acid molecule, for instance, as a hybridization probe or a polymerase chain reaction (PCR) primer. Uses of the nucleic acid molecules of the present invention that do not encode a polypeptide having TR2 receptor activity include, inter alia, (1) isolating a TR2 receptor gene or allelic or splice variants thereof in a cDNA library; (2) in situ hybridization (e.g., “FISH”) to metaphase chromosomal spreads to provide precise chromosomal location of a TR2 receptor gene, as described in Verma et al., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York (1988); and (3) Northern Blot analysis for detecting TR2 receptor mRNA expression in specific tissues.

Preferred, however, are nucleic acid molecules having sequences at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence shown in SEQ ID NO:25, FIG. 1A-1B (SEQ ID NO:1), FIG. 4A-4B (SEQ ID NO:4), or FIG. 7A-7B (SEQ ID NO:7) or to the nucleic acid sequence of the deposited cDNAs which do, in fact, encode a polypeptide having TR2 receptor activity.

Preferably, the polynucleotide fragments of the invention encode a polypeptide which demonstrates a TR2 functional activity. By “a polypeptide having TR2 receptor activity” is intended polypeptides exhibiting activity similar, but not necessarily identical, to an activity of the TR2 receptors of the present invention (e.g., complete (full-length) TR2 receptor polypeptides, mature TR2 receptor polypeptides, secreted TR2 receptor polypeptides, and soluble TR2 receptor polypeptides (e.g., having sequences contained in the extracellular domain of a TR2 receptor) as measured, for example, in a particular immunoassay or biological assay. For example, a TR2 receptor activity can routinely be measured by determining the ability of a TR2 receptor polypeptide to bind a TR2 receptor ligand (e.g., AIM II (International Publication No. WO 97/34911), Lymphotoxin-α, and the Herpes virus protein HSV1 gD). TR2 receptor activity can be measured by determining the ability of a polypeptide-Fc fusion protein to inhibit lymphocyte proliferation as described below in Example 6. TR2 receptor activity may also be measured by determining the ability of a polypeptide, such as cognate ligand which is free or expressed on a cell surface, to confer proliferatory activity in intact cells expressing the receptor.

Other methods will be known to the skilled artisan and are within the scope of the invention.

Of course, due to the degeneracy of the genetic code, one of ordinary skill in the art will immediately recognize that a large number of the nucleic acid molecules having a sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of the deposited cDNAs or the nucleic acid sequences shown in SEQ ID NO:25, FIG. 1A-1B (SEQ ID NO:1), FIG. 4A-4B (SEQ ID NO:4), or FIG. 7A-7B (SEQ ID NO:7) will encode polypeptides “having TR2 receptor activity.” In fact, since degenerate variants of any of these nucleotide sequences all encode the same polypeptide, this will be clear to the skilled artisan even without performing the above described comparison assay. It will be further recognized in the art that, for such nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide having TR2 protein activity. This is because the skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to significantly effect protein function (e.g., replacing one aliphatic amino acid with a second aliphatic amino acid).

For example, guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie, J. U. et al, “Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions,” Science 247:1306-1310 (1990), wherein the authors indicate that proteins are surprisingly tolerant of amino acid substitutions.

The present invention is further directed to polynucleotides comprising, or alternatively consisting of, fragments of the isolated nucleic acid molecules described herein. By a fragment of an isolated nucleic acid molecule having the nucleotide sequence of the deposited cDNAs or the nucleotide sequence shown in SEQ ID NO:25, FIG. 1A-1B (SEQ ID NO:1), FIG. 4A-4B (SEQ ID NO:4), or FIG. 7A-7B (SEQ ID NO:7) is intended fragments at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length which are useful as diagnostic probes and primers as discussed herein. In this context “about” includes the particularly recited value and values larger or smaller by several (5, 4, 3, 2, or 1) nucleotides.

Of course, larger fragments 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825 or 848 nt in length are also useful according to the present invention as are fragments corresponding to most, if not all, of the nucleotide sequences of the deposited cDNAs or as shown in SEQ ID NO:25, FIG. 1A-1B (SEQ ID NO:1), FIG. 4A-4B (SEQ ID NO:4), or FIG. 7A-7B (SEQ ID NO:7). By a fragment at least 20 nt in length, for example, is intended fragments which include 20 or more contiguous bases from the nucleotide sequences of the deposited cDNAs or the nucleotide sequences as shown in SEQ ID NO:25, FIG. 1A-1B (SEQ ID NO:1), FIG. 4A-4B (SEQ ID NO:4), or FIG. 7A-7B (SEQ ID NO:7).

Preferred nucleic acid fragments of the present invention include nucleic acid molecules encoding polypeptides comprising, or alternatively consisting of, the mature TR2-SV1 receptor (predicted to constitute amino acid residues from about 37 to about 185 in FIG. 4A-4B (amino acid residues 1 to 149 in SEQ ID NO:5)) and the complete TR2-SV2 receptor (predicted to constitute amino acid residues from about 1 to about 136 in FIG. 7A-7B (SEQ ID NO:8)). In this context “about” includes the particularly recited value and values larger or smaller by several (5, 4, 3, 2, or 1) amino acids.

As above with the leader sequence, the amino acid residues constituting the extracellular, transmembrane and intracellular domains have been predicted by computer analysis. Thus, as one of ordinary skill would appreciate, the amino acid residues constituting these domains may vary slightly (e.g., by about 1 to about 15 amino acid residues) depending on the criteria used to define each domain. In this context “about” includes the particularly recited value and values larger or smaller by several (5, 4, 3, 2, or 1) amino acids.

Preferred nucleic acid fragments of the present invention also include nucleic acid molecules encoding: a polypeptide comprising, or alternatively consisting of, the TR2 receptor protein of FIG. 1A-1B (SEQ ID NO:2) extracellular domain (predicted to constitute amino acid residues from about 37 to about 200 in FIG. 1A-1B (amino acid residues 1 to 164 in SEQ ID NO:2)); a polypeptide comprising, or alternatively consisting of, the TR2 receptor transmembrane domain (amino acid residues 201 to 225 in FIG. 1A-1B (amino acid residues 165 to 189 in SEQ ID NO:2)); a polypeptide comprising, or alternatively consisting of, the TR2 receptor intracellular domain (predicted to constitute amino acid residues from about 226 to about 283 in FIG. 1A-1B (amino acid residues 190 to 247 in SEQ ID NO:2)); and a polypeptide comprising, or alternatively consisting of, the TR2 receptor protein of FIG. 1A-1B (SEQ ID NO:2) extracellular and intracellular domains with all or part of the transmembrane domain deleted. In this context “about” includes the particularly recited value and values larger or smaller by several (5, 4, 3, 2, or 1) amino acids.

Preferred nucleic acid fragments of the present invention also include nucleic acid molecules encoding amino acid residues the extracellular domain of the TR2 protein having the amino acid sequence set out in SEQ ID NO:26, both with and without the associated leader sequence (amino acid residues −38 to 162 of SEQ ID NO:26 and amino acid residues 1 to 162 of SEQ ID NO:26, respectively).

Preferred nucleic acid fragments of the present invention also include nucleic acid molecules encoding epitope-bearing portions of the TR2 receptor proteins. In particular, such nucleic acid fragments of the present invention include nucleic acid molecules encoding: a polypeptide comprising, or alternatively consisting of, one, two, three, four, five or more amino acid sequences selected from amino acid residues from about 39 to about 70 in FIG. 1A-1B (amino acid residues 3 to 34 in SEQ ID NO:2); a polypeptide comprising, or alternatively consisting of, amino acid residues from about 106 to about 120 in FIG. 1 (amino acid residues 70 to 84 in SEQ ID NO:2); a polypeptide comprising, or alternatively consisting of, amino acid residues from about 142 to about 189 in FIG. 1A-1B (amino acid residues 106 to 153 in SEQ ID NO:2); a polypeptide comprising, or alternatively consisting of, amino acid residues from about 276 to about 283 in FIG. 1A-1B (amino acid residues 240 to 247 in SEQ ID NO:2); a polypeptide comprising, or alternatively consisting of, amino acid residues from about 39 to about 70 in FIG. 4A-4B (amino acid residues 3 to 34 in SEQ ID NO:5); amino acid residues from about 99 to about 136 in FIG. 4A-4B (amino acid residues 63 to 100 in SEQ ID NO:5); amino acid residues from about 171 to about 185 in FIG. 4A-4B (amino acid residues 135 to 149 in SEQ ID NO:5); amino acid residues from about 56 to about 68 in FIG. 7A-7B (SEQ ID NO:8); amino acid residues from about 93 to about 136 in FIG. 7A-7B (SEQ ID NO:8). In this context “about” includes the particularly recited value and values larger or smaller by several (5, 4, 3, 2, or 1) amino acids. The inventors have determined that the above polypeptide fragments are antigenic regions of the TR2 receptors. Methods for determining other such epitope-bearing portions of the TR2 proteins are described in detail below. Polypeptides encoded by these polynucleotides are also encompassed by the invention.

Representative examples of TR2 receptor polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 1 to 64, 65 to 100, 101 to 150, 151 to 200, 201 to 250, 225-265, 251 to 300, 301 to 350, 351 to 372, 373 to 450, 451 to 500, 501 to 550, 551 to 600, 601 to 650, 651 to 700, 701 to 750, 751 to 800, 801 to 850, 851 to 900, 901 to 950, 951 to 1000, 1001 to 1050, 1051 to 1100, 1070-1113, 1101 to 1150, 1151 to 1200, 1201 to 1250, 1251 to 1300, 1301 to 1350, 1351 to 1400, 1401 to 1450, 1451 to 1500, 1501 to 1550, 1551 to 1600, or 1601 to 1670, of SEQ ID NO:1, the cDNA contained in the deposited identified as ATCC Deposit No. 97059, or the complementary strand of any of these fragments. In this context “about” includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Polypeptides encoded by these polynucleotides are also encompassed by the invention.

Further representative examples of TR2 receptor polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 373 to 433, 373 to 450, 451 to 500, 501 to 550, 551 to 600, 601 to 650, 651 to 700, 701 to 750, 751 to 800, 801 to 850, 851 to 900, or 901 to 927 of SEQ ID NO:4, from about nucleotide 247 to 300, 301 to 350, 351 to 372, 373 to 450, 451 to 500, 501 to 550, 551 to 600, or 601 to 654 of SEQ ID NO:7, or the cDNA contained in the deposited identified as ATCC Deposit No. 97058 or 97057, or the complementary strand of any of these polynucleotides. In this context “about” includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. Polypeptides encoded by these polynucleotides are also encompassed by the invention.

It is believed one or more of the cysteine repeat regions of the TR2 receptor disclosed in FIG. 1A-1B are important for interactions between the TR2 receptor and its ligands (e.g., AIM II (International Publication No. WO 97/34911), Lymphotoxin-α, and the Herpes virus protein HSV1 glycoprotein D (gD)). Accordingly, specific embodiments of the invention are directed to polynucleotides encoding polypeptides which comprise, or alternatively consist of, the amino acid sequence of cysteine repeat region A, B, C, or D disclosed in FIG. 16 and described in Example 6. Additional embodiments of the invention are directed to polynucleotides encoding TR2 receptor polypeptides which comprise, or alternatively consist of, any combination of 1, 2, 3, or all 4 of cysteine repeat regions A-D disclosed in FIG. 16 and described in Example 6. Additional preferred embodiments of the invention are directed to polypeptides which comprise, or alternatively consist of, the TR2 receptor amino acid sequence of cysteine repeat region A, B, C, or D disclosed in FIG. 16 and described in Example 6. Additional embodiments of the invention are directed to TR2 receptor polypeptides which comprise, or alternatively consist of, any combination of 1, 2, 3, or all 4 of cysteine repeat regions A-D disclosed in FIG. 16 and described in Example 6.

In certain embodiments, polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequence encoding one, two, or all three of the cysteine-rich motifs described above. The present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence. Polypeptides encoded by these nucleic acids and/or polynucleotide sequences are also encompassed by the invention.

In another embodiment, the invention provides an isolated nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide which hybridizes under stringent hybridization conditions to one, two, or all three of the cysteine-rich motifs described above polynucleotides of the invention described above, or the complementary strand thereof. The meaning of the phrase “stringent conditions” as used herein is described infra.

Preferably, the polynucleotide fragments of the invention encode a polypeptide which demonstrates one or more TR2 receptor functional activities. By a polypeptide demonstrating a TR2 receptor “functional activity” is meant, a polypeptide capable of displaying one or more known functional activities associated with a full-length (complete) TR2 receptor protein. Such functional activities include, but are not limited to, biological activity (e.g., inhibition of B cell proliferation), antigenicity, immunogenicity (ability to generate antibody which binds to a TR2 receptor polypeptide), the ability to bind (or compete with a TR2 receptor polypeptide for binding) to an anti-TR2 receptor antibody, the ability to form multimers with TR2 receptor polypeptides of the invention, and ability to bind to a receptor or ligand for a TR2 receptor polypeptide (e.g., AIM II (International Publication No. WO 97/34911), Lymphotoxin-α, and the Herpes virus protein HSV1 glycoprotein D (gD)).

The functional activity of TR2 receptor polypeptides, and fragments, variants derivatives, and analogs thereof, can be assayed by various methods.

For example, in one embodiment where one is assaying for the ability to bind or compete with full-length TR2 receptor polypeptides for binding to anti-TR2 receptor antibody, various immunoassays known in the art can be used, including but not limited to, competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.

In another embodiment, where a TR2 receptor ligand is identified (e.g., AIM II (International Publication No. WO 97/34911), Lymphotoxin-α, and the Herpes virus protein HSV1 gD), or the ability of a polypeptide fragment, variant or derivative of the invention to multimerize is being evaluated, binding can be assayed, e.g., by means well-known in the art, such as, for example, reducing and non-reducing gel chromatography, protein affinity chromatography, and affinity blotting. See generally, Phizicky, E., et al., Microbiol. Rev. 59:94-123 (1995). In another embodiment, physiological correlates of TR2 receptor binding to its substrates (signal transduction) can be assayed.

In addition, assays described herein (see, e.g., Examples 6 and 8 and otherwise known in the art may routinely be applied to measure the ability of TR2 receptor polypeptides and fragments, variants derivatives and analogs thereof to elicit TR2 receptor related biological activity (e.g., inhibition of B cell proliferation in vitro or in vivo). Other methods will be known to the skilled artisan and are within the scope of the invention.

In another aspect, the invention provides isolated nucleic acid molecules comprising, or alternatively consisting of, polynucleotides which hybridizes under stringent hybridization conditions to a portion of the polynucleotide of one of the nucleic acid molecules of the invention described above, for instance, the complement of a polynucleotide fragment described herein, or the cDNAs contained in ATCC Deposits 97059, 97058 and 97057. By “stringent hybridization conditions” is intended overnight incubation at 42° C. in a solution comprising, or alternatively consisting of 50% formamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC at about 65° C.

By a polynucleotide which hybridizes to a “portion” of a polynucleotide is intended a polynucleotide (either DNA or RNA) hybridizing to at least about 15 nucleotides (nt), and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably about 30-70 nt of the reference polynucleotide. In this context “about” includes the particularly recited value and values larger or smaller by several (5, 4, 3, 2, or 1) nucleotide. These are useful as diagnostic probes and primers as discussed above and in more detail below.

By a portion of a polynucleotide of “at least 20 nt in length,” for example, is intended 20 or more contiguous nucleotides from the nucleotide sequence of the reference polynucleotide (e.g., the deposited cDNAs or the nucleotide sequences as shown in SEQ ID NO:25, FIG. 1A-1B (SEQ ID NO:1), FIG. 4A-4B (SEQ ID NO:4), or FIG. 7A-7B (SEQ ID NO:7)).

Of course, a polynucleotide which hybridizes only to a poly A sequence (such as the 3′ terminal poly(A) tract of the TR2 receptor cDNA sequences shown in SEQ ID NO:25, FIG. 1A-1B (SEQ ID NO:1), FIG. 4A-4B (SEQ ID NO:4), or FIG. 7A-7B (SEQ ID NO:7)), or to a complementary stretch of T (or U) resides, would not be included in a polynucleotide of the invention used to hybridize to a portion of a nucleic acid of the invention, since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone generated from an oligo-dT primed cDNA library).

As indicated, nucleic acid molecules of the present invention which encode TR2 polypeptides may include, but are not limited to those encoding the amino acid sequences of the mature polypeptides, by itself, the coding sequence for the mature polypeptides and additional sequences, such as those encoding the about 36 amino acid leader or secretory sequences, such as pre-, or pro- or prepro-protein sequences; the coding sequence of the mature polypeptides, with or without the aforementioned additional coding sequences, together with additional, non-coding sequences, including for example, but not limited to introns and non-coding 5′ and 3′ sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing, including splicing and polyadenylation signals, for example—ribosome binding and stability of mRNA; an additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities. Thus, the sequence encoding the polypeptides may be fused to a marker sequence, such as a sequence encoding a peptide which facilitates purification of the fused polypeptide. In certain preferred embodiments of this aspect of the invention, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. The “HA” tag is another peptide useful for purification which corresponds to an epitope derived from the influenza hemagglutinin protein, which has been described by Wilson et al., Cell 37: 767 (1984). As discussed below, other such fusion proteins include the TR2 receptors fused to IgG-Fc at the N- or C-terminus.

The present invention further relates to variants of the nucleic acid molecules of the present invention, which encode portions, analogs or derivatives of the TR2 receptors. Variants may occur naturally, such as a natural allelic variant. By an “allelic variant” is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985).

Non-naturally occurring variants may be produced using art-known mutagenesis techniques, which include, but are not limited to: oligonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis, site directed mutagenesis (see, e.g., Carter et al, Nucl. Acids Res. 13:4331 (1986); and Zoller et al., Nucl. Acids Res. 10: 6487 (1982)), cassette mutagenesis (see, e.g., Wells et al., Gene 34:315 (1985)), restriction selection mutagenesis (see, e.g., Wells et al., Philos. Trans. R. Soc. London Ser.A 317:415 (1986)).

Such variants include those produced by nucleotide substitutions, deletions or additions, which may involve one or more nucleotides. The variants may be altered in coding regions, non-coding regions, or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the TR2 receptors or portions thereof. Also especially preferred in this regard are conservative substitutions.

Vectors and Host Cells

The present invention also relates to vectors which include the isolated DNA molecules of the present invention, host cells which are genetically engineered with the recombinant vectors, and the production of TR2 polypeptides or fragments thereof by recombinant techniques.

The polynucleotides may be joined to a vector containing a selectable marker for propagation in a host. Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.

The DNA insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp and fac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters will be known to the skilled artisan. The expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs will preferably include a translation initiating at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.

As indicated, the expression vectors will preferably include at least one selectable marker. Such markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria. Representative examples of appropriate heterologous hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS and Bowes melanoma cells; and plant cells. Appropriate culture mediums and conditions for the above-described host cells are known in the art.

Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. Other suitable vectors will be readily apparent to the skilled artisan.

Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986).

The polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art. A preferred fusion protein comprises a heterologous region from immunoglobulin that is useful to solubilize proteins. For example, EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobin molecules together with another human protein or part thereof. In many cases, the Fc part in a fusion protein is thoroughly advantageous for use in therapy and diagnosis and thus results, for example, in improved pharmacokinetic properties (EP-A 0232262). On the other hand, for some uses it would be desirable to be able to delete the Fc part after the fusion protein has been expressed, detected and purified in the advantageous manner described. This is the case when Fc portion proves to be a hindrance to use in therapy and diagnosis, for example when the fusion protein is to be used as antigen for immunizations. In drug discovery, for example, human proteins, such as, human hIL-5 receptor has been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. See, D. Bennett et al., Journal of Molecular Recognition, Vol. 8:52-58 (1995) and K. Johanson et al., The Journal of Biological Chemistry, Vol. 270, No. 16:9459-9471 (1995).

TR2 receptors can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification. Polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.

In addition to encompassing host cells containing the vector constructs discussed herein, the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., the TR2 receptor coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with TR2 receptor polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous TR2 receptor polynucleotides. For example, techniques known in the art may be used to operably associate heterologous control regions (e.g., promoter and/or enhancer) and endogenous TR2 receptor polynucleotide sequences via homologous recombination (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997; International Publication Number WO 96/29411, published Sep. 26, 1996; International Publication Number WO 94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci. U.S.A. 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989), the disclosures of each of which are incorporated by reference in their entireties).

TR2 Polypeptides and Fragments

The invention further provides isolated TR2 polypeptides having the amino acid sequence encoded by the deposited cDNAs, or the amino acid sequence shown in SEQ ID NO:26, FIG. 1A-1B (SEQ ID NO:2), FIG. 4A-4B (SEQ ID NO:5), or FIG. 7A-7B (SEQ ID NO:8), or a peptide or polypeptide comprising, or alternatively consisting of, a portion of the above polypeptides.

The polypeptides of this invention may be membrane bound or may be in a soluble circulating form. Soluble peptides are defined by amino acid sequence wherein the sequence comprises, or alternatively consists of, the polypeptide sequence lacking the transmembrane domain. One example of such a soluble form of the TR2 receptor is the TR2-SV1 splice variant which has a secretory leader sequence but lacks both the intracellular and transmembrane domains. Thus, the TR2-SV1 receptor protein appears to be secreted in a soluble form from cells which express this protein.

The polypeptides of the present invention may exist as a membrane bound receptor having a transmembrane region and an intra- and extracellular region or they may exist in soluble form wherein the transmembrane domain is lacking. One example of such a form of the TR2 receptor is the TR2 receptor shown in FIG. 1A-1B (SEQ ID NO:2) which contains, in addition to a leader sequence, transmembrane, intracellular and extracellular domains. Thus, this form of the TR2 receptor appears to be localized in the cytoplasmic membrane of cells which express this protein.

The polypeptides of the present invention also include the polypeptide encoded by the deposited cDNAs including the leader; the polypeptide encoded by the deposited the cDNAs minus the leader (i.e., the mature protein); the polypeptides of SEQ ID NO:26, FIG. 1A-1B (SEQ ID NO:2) or FIG. 4A-4B (SEQ ID NO:5) including the leader; the polypeptides of SEQ ID NO:26, FIG. 1A-1B (SEQ ID NO:2) or FIG. 4A-4B (SEQ ID NO:5) including the leader but minus the N-terminal methionine; the polypeptides of SEQ ID NO:26, FIG. 1A-1B (SEQ ID NO:2) or FIG. 4A-4B (SEQ ID NO:5) minus the leader; the polypeptide of FIG. 7A-7B (SEQ ID NO:8); the extracellular domain, the transmembrane domain, and the intracellular domain of the TR2 receptor shown in SEQ ID NO:26 or FIG. 1A-1B (SEQ ID NO:2); and polypeptides which are at least 80% identical, more preferably at least 85%, 90%, 92% or 95% identical, still more preferably at least 96%, 97%, 98% or 99% identical to the polypeptides described above, and also include portions of such polypeptides with at least 30 amino acids and more preferably at least 50 amino acids.

By a polypeptide having an amino acid sequence at least, for example, 95% “identical” to a reference amino acid sequence of a TR2 polypeptide is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of a TR2 receptor. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence, up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence. These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence shown in SEQ ID NO:26, FIG. 1A-1B (SEQ ID NO:2), FIG. 4A-4B (SEQ ID NO:5), or FIG. 7A-7B (SEQ ID NO:8) or to the amino acid sequence encoded by one of the deposited cDNAs can be determined conventionally using known computer programs such the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711). When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference amino acid sequence and that gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence are allowed.

In a specific embodiment, the identity between a reference (query) sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, is determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)). Preferred parameters used in a FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, Window Size=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject amino acid sequence, whichever is shorter. According to this embodiment, if the subject sequence is shorter than the query sequence due to N- or C-terminal deletions, not because of internal deletions, a manual correction is made to the results to take into consideration the fact that the FASTDB program does not account for N- and C-terminal truncations of the subject sequence when calculating global percent identity. For subject sequences truncated at the N- and C-termini, relative to the query sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence. A determination of whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of this embodiment. Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N- and C-terminal residues of the subject sequence. For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity. The deletion occurs at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not show a matching/alignment of the first 10 residues at the N-teRminus. The 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C-termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%. In another example, a 90 residue subject sequence is compared with a 100 residue query sequence. This time the deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are made for the purposes of this embodiment.

It will be recognized in the art that some amino acid sequences of the TR2 receptors can be varied without significant effect to the structure or function of the protein. If such differences in sequence are contemplated, it should be remembered that there will be critical areas on the protein which determine activity. Thus, the invention further includes variations of the TR2 receptors which show substantial TR2 receptor activity or which include regions of TR2 proteins such as the protein portions discussed below. Such mutants include deletions, insertions, inversions, repeats, and type substitutions. Guidance concerning which amino acid changes are likely to be phenotypically silent can be found in Bowie, J. U., et al., “Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions,” Science 247:1306-1310 (1990).

Thus, the fragment, derivative or analog of the polypeptides of SEQ ID NO:26, FIG. 1A-1B (SEQ ID NO:2), FIG. 4A-4B (SEQ ID NO:5), and FIG. 7A-7B (SEQ ID NO:8), or that encoded by the deposited cDNAs, may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as an IgG Fc fusion region peptide or leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein. Polynucleotides encoding these fragments, derivatives or analogs are also encompassed by the invention.

Of particular interest are substitutions of charged amino acids with another charged amino acid and with neutral or negatively charged amino acids. The latter results in proteins with reduced positive charge to improve the characteristics of the TR2 proteins. The prevention of aggregation is highly desirable. Aggregation of proteins not only results in a loss of activity but can also be problematic when preparing pharmaceutical formulations, because they can be immunogenic. (Pinckard et al., Clin Exp. Immunol 2:331-340 (1967); Robbins et al., Diabetes 36:838-845 (1987); Cleland et al. Crit. Rev. Therapeutic Drug Carrier Systems 10:307-377 (1993)).

The replacement of amino acids can also change the selectivity of binding to cell surface receptors. Ostade et al., Nature 361:266-268 (1993) describes certain mutations resulting in selective binding of TNF-α to only one of the two known types of TNF receptors. Thus, the TR2 receptors of the present invention may include one or more amino acid substitutions, deletions or additions, either from natural mutations or human manipulation.

As indicated, changes are preferably of a minor nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity of the protein (see Table I).

TABLE I
CONSERVATIVE AMINO ACID SUBSTITUTIONS.
Aromatic Phenylalanine
Tryptophan
Tyrosine
Hydrophobic Leucine
Isoleucine
Valine
Polar Glutamine
Asparagine
Basic Arginine
Lysine
Histidine
Acidic Aspartic Acid
Glutamic Acid
Small Alanine
Serine
Threonine
Methionine
Glycine

Amino acids in the TR2 proteins of the present invention that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity such as receptor binding or in vitro, or in vitro proliferative activity. Sites that are critical for ligand-receptor binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et at, J. Mol. Biol. 224:899-904 (1992) and de Vos et al. Science 255:306-312 (1992)).

The polypeptides of the present invention are preferably provided in an isolated form. By “Isolated polypeptide”, is intended a polypeptide removed from its native environment. Thus, a polypeptide produced and contained within a recombinant host cell would be considered “isolated” for purposes of the present invention. Also intended as an “isolated polypeptide” are polypeptides that have been purified, partially or substantially, from a recombinant host. For example, recombinantly produced versions of the TR2 receptors can be substantially purified by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988).

The polypeptides of the present invention have uses which include, but are not limited to, as sources for generating antibodies that bind the polypeptides of the invention, and as molecular weight markers on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art.

TR2 polypeptides of the invention can also inhibit mixed lymphocyte reactions (MLRs). As discussed below in Example 6, TR2 polypeptides inhibit three-way MLRs. An additional method for performing three-way MLRs is discussed in Harrop et al, Jour. Immunol. 161:1786-1794 (1998), which incorporated herein by reference.

The present application is also directed to proteins containing polypeptides at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the TR2 receptor polypeptide sequence set forth herein as n1-m1, n2-m2, n3-m3, n4-m4, and/or n5-m5. In preferred embodiments, the application is directed to proteins containing polypeptides at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to polypeptides having the amino acid sequence of the specific TR2 receptor N- and C-terminal deletions recited herein. Polynucleotides encoding these polypeptides are also encompassed by the invention.

In certain preferred embodiments, TR2 receptor proteins of the invention comprise, or alternatively consist of, fusion proteins as described above wherein the TR2 receptor polypeptides are those described as n1-m1, n2-m2, n3-m3, n4-m4, and/or n5-m5 herein. In preferred embodiments, the application is directed to nucleic acid molecules at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequences encoding polypeptides having the amino acid sequence of the specific N- and C-terminal deletions recited herein. Polynucleotides encoding these polypeptides are also encompassed by the invention.

As mentioned above, even if deletion of one or more amino acids from the N-terminus of a protein results in modification of loss of one or more biological functions of the protein, other biological activities may still be retained. Thus, the ability of shortened TR2 muteins to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptides generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the N-terminus. Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a TR2 mutein with a large number of deleted N-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six TR2 amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the TR2 amino acid sequence shown in FIG. 1A-1B (i.e., SEQ ID NO:2), up to the glycine residue at position number 278 and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues n1-283 of FIG. 1A-1B (SEQ ID NO:2), where n1 is an integer in the range of 2 to 278. Polynucleotides encoded by these polypeptides are also encompassed by the invention.

More in particular, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues of E-2 to H-283; P-3 to H-283; P-4 to H-283; G-5 to H-283; D-6 to H-283; W-7 to H-283; G-8 to H-283; P-9 to H-283; P-10 to H-283; P-11 to H-283; W-12 to H-283; R-13 to H-283; S-14 to H-283; T-15 to H-283; P-16 to H-283; K-17 to H-283; T-18 to H-283; D-19 to H-283; V-20 to H-283; L-21 to H-283; R-22 to H-283; L-23 to H-283; V-24 to H-283; L-25 to H-283; Y-26 to H-283; L-27 to H-283; T-28 to H-283; F-29 to H-283; L-30 to H-283; G-31 to H-283; A-32 to H-283; P-33 to H-283; C-34 to H-283; Y-35 to H-283; A-36 to H-283; P-37 to H-283; A-38 to H-283; L-39 to H-283; P-40 to H-283; S-41 to H-283; C-42 to H-283; K-43 to H-283; E-44 to H-283; D-45 to H-283; E-46 to H-283; Y-47 to H-283; P-48 to H-283; V-49 to H-283; G-50 to H-283; S-51 to H-283; E-52 to H-283; C-53 to H-283; C-54 to H-283; P-55 to H-283; K-56 to H-283; C-57 to H-283; S-58 to H-283; P-59 to H-283; G-60 to H-283; Y-61 to H-283; R-62 to H-283; V-63 to H-283; K-64 to H-283; E-65 to H-283; A-66 to H-283; C-67 to H-283; G-68 to H-283; E-69 to H-283; L-70 to H-283; T-71 to H-283; G-72 to H-283; T-73 to H-283; V-74 to H-283; C-75 to H-283; E-76 to H-283; P-77 to H-283; C-78 to H-283; P-79 to H-283; P-80 to H-283; G-81 to H-283: T-82 to H-283; Y-83 to H-283; 1-84 to H-283; A-85 to H-283; H-86 to H-283; L-87 to H-283; N-88 to H-283; G-89 to H-283; L-90 to H-283; S-91 to H-283; K-92 to H-283; C-93 to H-283; L-94 to H-283; Q-95 to H-283; C-96 to H-283; Q-97 to H-283; M-98 to H-283; C-99 to H-283; D-100 to H-283; P-101 to H-283; A-102 to H-283; M-103 to H-283; G-104 to H-283; L-105 to H-283; R-106 to H-283; A-107 to H-283; S-108 to H-283; R-109 to H-283; N-110 to H-283; C-111 to H-283; S-112 to H-283; R-113 to H-283; T-114 to H-283; E-115 to H-283; N-116 to H-283; A-117 to H-283; V-118 to H-283; C-119 to H-283; G-120 to H-283; C-121 to H-283; S-122 to H-283; P-123 to H-283; G-124 to H-283; H-125 to H-283; F-126 to H-283; C-127 to H-283; 1-128 to H-283; V-129 to H-283; Q-130 to H-283; D-103 to H-283; G-132 to H-283; D-133 to H-283; H-134 to H-283; C-135 to H-283; A-136 to H-283; A-137 to H-283; C-138 to H-283; R-139 to H-283; A-140 to H-283; Y-141 to H-283; A-142 to H-283; T-143 to H-283; S-144 to H-283; S-145 to H-283; P-146 to H-283; G-147 to H-283; Q-148 to H-283; R-149 to H-283; V-150 to H-283; Q-151 to H-283; K-152 to H-283; G-153 to H-283; G-154 to H-283; T-155 to H-283; E-156 to H-283; S-157 to H-283; Q-158 to H-283; D-159 to H-283; T-160 to H-283; L-161 to H-283; C-162 to H-283; Q-163 to H-283; N-164 to H-283; C-165 to H-283; P-166 to H-283; P-167 to H-283; G-168 to H-283; T-169 to H-283; F-170 to H-283; S-171 to H-283; P-172 to H-283; N-173 to H-283; G-174 to H-283; T-175 to H-283; L-176 to H-283; E-177 to H-283; E-178 to H-283; C-179 to H-283; Q-180 to H-283; H-181 to H-283; Q-182 to H-283; T-183 to H-283; K-184 to H-283; C-185 to H-283; S-186 to H-283; W-187 to H-283; L-188 to H-283; V-189 to H-283; T-190 to H-283; K-191 to H-283; A-192 to H-283; G-193 to H-283; A-194 to H-283; G-195 to H-283; T-196 to H-283; S-197 to H-283; S-198 to H-283; S-199 to H-283; H-200 to 1H-283; W-201 to H-283; V-202 to H-283; W-203 to H-283; W-204 to H-283; F-205 to H-283; L-206 to H-283; S-207 to H-283; G-208 to H-283; S-209 to H-283; L-210 to H-283; V-211 to H-283; I-212 to H-283; V-213 to H-283; I-214 to H-283; V-215 to H-283; C-216 to H-283; S-217 to H-283; T-218 to H-283; V-219 to H-283; G-220 to H-283; L-221 to H-283; 1-222 to H-283; 1-223 to H-283; C-224 to H-283; V-225 to H-283; K-226 to H-283; R-227 to H-283; R-228 to H-283; K-229 to H-283; P-230 to H-283; R-231 to H-283; G-232 to H-283; D-233 to H-283; V-234 to H-283; V-235 to H-283; K-236 to H-283; V-237 to H-283; 1-238 to H-283; V-239 to H-283; S-240 to H-283; V-241 to H-283; Q-242 to H-283; R-243 to H-283; K-244 to H-283; R-245 to H-283; Q-246 to H-283; E-247 to H-283; A-248 to H-283; E-249 to H-283; G-250 to H-283; E-251 to H-283; A-252 to H-283; T-253 to H-283; V-254 to H-283; I-255 to H-283; E-256 to H-283; A-257 to H-283; L-258 to H-283; Q-259 to H-283; A-260 to H-283; P-261 to H-283; P-262 to H-283; D-263 to H-283; V-264 to H-283; T-265 to H-283; T-266 to H-283; V-267 to H-283; A-268 to H-283; V-269 to H-283; E-270 to H-283; E-271 to H-283; T-272 to H-283; I-273 to H-283; P-274 to H-283; S-275 to H-283; F-276 to H-283; T-277 to H-283; and G-278 to H-283 of the TR2 sequence shown in FIG. 1A-1B. The present invention is also directed to nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequences encoding the polypeptides described above. The present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence. Polypeptides encoded by these polynucleotide sequences are also encompassed by the invention.

Also as mentioned above, even if deletion of one or more amino acids from the C-terminus of a protein results in modification of loss of one or more biological functions of the protein, other biological activities may still be retained. Thus, the ability of the shortened TR2 mutein to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a TR2 mutein with a large number of deleted C-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six TR2 amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the TR2 polypeptide shown in FIG. 1A-1B (SEQ ID NO:2), up to the aspartic acid residue at position number 6, and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues 1-m1 of FIG. 1A-1B (i.e., SEQ ID NO:2), where m1 is an integer in the range of 6 to 282. Polynucleotides encoded by these polypeptides are also encompassed by the invention.

More in particular, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues M-1 to N-282; M-1 to P-281; M-1 to S-280; M-1 to R-279; M-1 to G-278; M-1 to T-277; M-1 to F-276; M-1 to S-275; M-1 to P-274; M-1 to I-273; M-1 to T-272; M-1 to E-271; M-1 to E-270; M-1 to V-269; M-1 to A-268; M-1 to V-267; M-1 to T-266; M-1 to T-265; M-1 to V-264; M-1 to D-263; M-1 to P-262; M-1 to P-261; M-1 to A-260; M-1 to Q-259; M-1 to L-258; M-1 to A-257; M-1 to E-256; M-1 to 1-255; M-1 to V-254; M-1 to T-253; M-1 to A-252; M-1 to E-251; M-1 to G-250; M-1 to E-249; M-1 to A-248; M-1 to E-247; M-1 to Q-246; M-1 to R-245; M-1 to K-244; M-1 to R-243; M-1 to Q-242; M-1 to V-241; M-1 to S-240; M-1 to V-239; M-1 to 1-238; M-1 to V-237. M-1 to K-236; M-1 to V-235; M-1 to V-234; M-1 to D-233; M-1 to G-232; M-1 to R-231; M-1 to P-230; M-1 to K-229; M-1 to R-228; M-1 to R-227; M-1 to K-226; M-1 to V-225; M-1 to C-224; M-1 to 1-223; M-1 to I-222; M-1 to L-221; M-1 to G-220; M-1 to V-219 M-1 to T-218; M-1 to S-217; M-1 to C-216; M-1 to V-215; M-1 to 1-214; M-1 to V-213; M-1 to S-212; M-1 to V-211; M-1 to L-215; M-1 to S-209; M-1 to 0-208; M-1 to S-207; M-1 to L-206; M-1 to F-205; M-1 to W-204; M-1 to W-203; M-1 to V-202; M-1 to W-201; M-1 to H-200; M-1 to S-199; M-1 to S-198; M-1 to S-197; M-1 to T-196; M-1 to G-195; M-1 to A-194; M-1 to G-193; M-1 to A-192; M-1 to K-191; M-1 to T-190; M-1 to V-189; M-1 to L-188; M-1 to W-187; M-1 to S-186; M-1 to C-185; M-1 to K-184; M-1 to T-183; M-1 to Q-182; M-1 to H-181; M-1 to Q-180; M-1 to C-179; M-1 to E-178; M-1 to E-177; M-1 to L-176; M-1 to T-175; M-1 to 0-174; M-1 to N-173; M-1 to P-172; M-1 to S-171; M-1 to F-170; M-1 to T-169M-1 to M-168; M-1 to P-167 M-1 to P-166 M-1 to C-165; M-1 to N-164; M-1 to Q-163; M-1 to C-162; M-1 to L-161; M-1 to T-160; M-1 to D-159; M-1 to Q-158; M-1 to S-157; M-1 to E-156; M-1 to T-155; M-1 to D-154; M-1 to G-153; M-1 to K-152; M-1 to Q-151; M-1 to V-150; M-1 to R-149; M-1 to Q-148; M-1 to G-147; M-1 to P-146; M-1 to S-145; M-1 to S-144; M-1 to T-143; M-1 to A-142; M-1 to Y-141; M-1 to A-140; M-1 to R-139; M-1 to C-138; M-1 to A-137; M-1 to A-136; M-1 to C-135; M-1 to H-134; M-1 to D-133; M-1 to G-132; M-1 to D-131; M-1 to Q-130; M-1 to V-129; M-1 to 1-128; M-1 to C-127; M-1 to F-126; M-1 to H-125; M-1 to G-124; M-1 to P-123; M-1 to S-122; M-1 to C-121; M-1 to G-120; M-1 to C-119; M-1 to V-118; M-1 to A-117; M-1 to N-116; M-1 to E-115; M-1 to T-114; M-1 to R-113; M-1 to S-112; M-1 to C-111; M-1 to N-110; M-1 to R-109; M-1 to S-108; M-1 to A-107; M-1 to R-106; M-1 to L-105; M-1 to G-104; M-1 to M-103; M-1 to A-102; M-1 to P-101; M-1 to D-100; M-1 to C-99; M-1 to M-98; M-1 to Q-97; M-1 to C-96; M-1 to Q-95; M-1 to L-94; M-1 to C-93; M-1 to K-92; M-1 to S-91; M-1 to L-90; M-1 to G-89; M-1 to N-88; M-1 to L-87; M-1 to H-86; M-1 to A-85; M-1 to 1-84; M-1 to Y-83; M-1 to T-82; M-1 to G-81; M-1 to P-80; M-1 to P-79; M-1 to C-78; M-1 to P-77; M-1 to E-76; M-1 to C-75; M-1 to V-74; M-1 to T-73; M-1 to G-72; M-1 to T-71; M-1 to L-70; M-1 to E-69; M-1 to G-68; M-1 to C-67; M-1 to A-66; M-1 to E-65; M-1 to K-64; M-1 to V-63; M-1 to R-62; M-1 to Y-61; M-1 to G-60; M-1 to P-59; M-1 to S-58; M-1 to C-57; M-1 to K-56; M-1 to P-55; M-1 to C-54; M-1 to C-53; M-1 to E-52; M-1 to S-51; M-1 to G-50; M-1 to V-49; M-1 to P-48; M-1 to Y-47; M-1 to E-46; M-1 to D-45; M-1 to E-44; M-1 to K-43; M-1 to C-42; M-1 to S-41; M-1 to P-40; M-1 to L-39; M-1 to A-38; M-1 to P-37; M-1 to A-36; M-1 to Y-35; M-1 to C-34; M-1 to P-33; M-1 to A-32; M-1 to G-31; M-1 to L-30; M-1 to F-29; M-1 to T-28; M-1 to L-27; M-1 to Y-26; M-1 to L-25; M-1 to V-24; M-1 to L-23; M-1 to R-22; M-1 to L-21; M-1 to V-20; M-1 to D-19; M-1 to T-18; M-1 to K-17; M-1 to P-16; M-1 to T-15; M-1 to S-14; M-1 to R-13; M-1 to W-12; M-1 to P-11; M-1 to P-10; M-1 to P-9; M-1 to G-8; M-1 to W-7; and M-1 to D-6 of the sequence of the TR2 sequence shown in FIG. 1A-1B. The present invention is also directed to nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequences encoding the polypeptides described above. The present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence. Polypeptides encoded by these polynucleotide sequences are also encompassed by the invention.

The invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini of a TR2 polypeptide, which may be described generally as having residues n1-m1 of FIG. 1A-1B (i.e., SEQ ID NO:2), where n1 and m1 are integers as described above. Polynucleotides encoded by these polypeptides are also encompassed by the invention.

Also mentioned above, even if deletion of one or more amino acids from the N-terminus of a protein results in modification of loss of one or more biological functions of the protein, other biological activities may still be retained. Thus, the ability of shortened TR2-SV1 muteins to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptides generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the N-terminus. Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a TR2-SV1 mutein with a large number of deleted N-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six TR2-SV1 amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the TR2-SV1 amino acid sequence shown in FIG. 4A-4B (i.e., SEQ ID NO:5), up to the threonine residue at position number 180 and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues n2-185 of FIG. 4A-4B (SEQ ID NO:5), where n2 is an integer in the range of 2 to 180, and 180 is the position of the first residue from the N-terminus of the complete TR2-SV1 polypeptide believed to be required for at least immunogenic activity of the TR2-SV1 polypeptide. Polynucleotides encoded by these polypeptides are also encompassed by the invention.

More in particular, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues of E-2 to A-185; P-3 to A-185; P-4 to A-185; G-5 to A-185; D-6 to A-185; W-7 to A-185; G-8 to A-185; P-9 to A-185; P-10 to A-185; P-11 to A-185; W-12 to A-185; R-13 to A-185; S-14 to A-185; T-15 to A-185; P-16 to A-185; R-17 to A-185; T-18 to A-185; D-19 to A-185; V-20 to A-185; L-21 to A-185; R-22 to A-185; L-23 to A-185; V-24 to A-185; L-25 to A-185; Y-26 to A-185; L-27 to A-185; T-28 to A-185; F-29 to A-185; L-30 to A-185; G-31 to A-185; A-32 to A-185; P-33 to A-185; C-34 to A-185; Y-35 to A-185; A-36 to A-185; P-37 to A-185; A-38 to A-185; L-39 to A-185; P-40 to A-185; S-41 to A-185; C-42 to A-185; K-43 to A-185; E-44 to A-185; D-45 to A-185; E-46 to A-185; Y-47 to A-185; P-48 to A-185; V-49 to A-185; G-50 to A-185; S-51 to A-185; E-52 to A-185; C-53 to A-185; C-54 to A-185; P-55 to A-185; K-56 to A-185; C-57 to A-185; S-58 to A-185; P-59 to A-185; G-60 to A-185; Y-61 to A-185; R-62 to A-185; V-63 to A-185; K-64 to A-185; E-65 to A-185; A-66 to A-185; C-67 to A-185; G-68 to A-185; E-69 to A-185; L-70 to A-185; T-71 to A-185; G-72 to A-185; T-73 to A-185; V-74 to A-185; C-75 to A-185; E-76 to A-185; P-77 to A-185; C-78 to A-185; P-79 to A-185; P-80 to A-185; G-81 to A-185; T-82 to A-185; Y-83 to A-185; 1-84 to A-185; A-85 to A-185; H-86 to A-185; L-87 to A-185; N-88 to A-185; G-89 to A-185; L-90 to A-185; S-91 to A-185; K-92 to A-185; C-93 to A-185; L-94 to A-185; Q-95 to A-185; C-96 to A-185; Q-97 to A-185; M-98 to A-185; C-99 to A-185; D-100 to A-185; P-101 to A-185; D-102 to A-185; 1-103 to A-185; G-104 to A-185; S-105 to A-185; P-106 to A-185; C-107 to A-185; D-108 to A-185; L-109 to A-185; R-110 to A-185; G-111 to A-185; R-112 to A-185; G-113 to A-185; H-114 to A-185; L-115 to A-185; E-116 to A-185; A-117 to A-185; G-118 to A-185; A-119 to A-185; H-120 to A-185; L-121 to A-185; S-122 to A-185; P-123 to A-185; G-124 to A-185; R-125 to A-185; Q-126 to A-185; K-127 to A-185; G-128 to A-185; E-129 to A-185; P-130 to A-185; D-131 to A-185; P-132 to A-185; E-133 to A-185; V-134 to A-185; A-135 to A-185; F-136 to A-185; E-137 to A-185; S-138 to A-185; L-139 to A-185; S-140 to A-185; A-141 to A-185; E-142 to A-185; P-143 to A-185; V-144 to A-185; H-145 to A-185; A-146 to A-185; A-147 to A-185; N-148 to A-185; G-149 to A-185; S-150 to A-185; V-151 to A-185; P-152 to A-185; L-153 to A-185; E-154 to A-185; P-155 to A-185; H-156 to A-185; A-157 to A-185; R-158 to A-185; L-159 to A-185; S-160 to A-185; M-161 to A-185; A-162 to A-185; S-163 to A-185; A-164 to A-185; P-165 to A-185; C-166 to A-185; G-167 to A-185; Q-168 to A-185; A-169 to A-185; G-170 to A-185; L-171 to A-185; H-172 to A-185; L-173 to A-185; R-174 to A-185; D-175 to A-185; R-176 to A-185; A-177 to A-185; D-178 to A-185; G-179 to A-185; and T-180 to A-185 of the TR2-SV1 sequence shown in FIG. 4A-4B. The present invention is also directed to nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequences encoding the polypeptides described above. The present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence. Polypeptides encoded by these polynucleotide sequences are also encompassed by the invention.

Also as mentioned above, even if deletion of one or more amino acids from the C-terminus of a protein results in modification of loss of one or more biological functions of the protein, other biological activities may still be retained. Thus, the ability of the shortened TR2-SV1 mutein to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a TR2-SV1 mutein with a large number of deleted C-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six TR2-SV1 amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the TR2-SV1 polypeptide shown in FIG. 4A-4B (SEQ ID NO:5), up to the aspartic acid residue at position number 6, and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues 1-m2 of FIG. 4A-4B (i.e., SEQ ID NO:5), where m2 is an integer in the range of 6 to 184. Polynucleotides encoded by these polypeptides are also encompassed by the invention.

More in particular, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues M-1 to R-184; M-1 to G-183; M-1 to G-182; M-1 to P-181; M-1 to T-180; M-1 to G-179; M-1 to D-178; M-1 to A-177; M-1 to R-176; M-1 to D-175; M-1 to R-174; M-1 to L-173; M-1 to H-172; M-1 to L-171; M-1 to G-170; M-1 to A-169; M-1 to Q-168; M-1 to G-167; M-1 to C-166; M-1 to P-165; M-1 to A-164; M-1 to S-163; M-1 to A-162; M-1 to M-161; M-1 to S-160; M-1 to L-159; M-1 to R-158; M-1 to A-157; M-1 to H-156; M-1 to P-155; M-1 to E-154; M-1 to L-153; M-1 to P-152; M-1 to V-151; M-1 to S-150; M-1 to G-149; M-1 to N-148; M-1 to A-147; M-1 to A-146; M-1 to H-145; M-1 to V-144; M-1 to P-143; M-1 to E-142; M-1 to A-141; M-1 to S-140; M-1 to L-139; M-1 to S-138; M-1 to E-137; M-1 to F-136; M-1 to A-135; M-1 to V-134; M-1 to E-133; M-1 to P-132; M-1 to D-131; M-1 to P-130; M-1 to E-129; M-1 to G-128; M-1 to K-127; M-1 to Q-126; M-1 to R-125; M-1 to G-124; M-1 to P-123; M-1 to S-122; M-1 to L-121; M-1 to H-120; M-1 to A-119; M-1 to G-118; M-1 to A-117; M-1 to E-116; M-1 to L-115; M-1 to H-114; M-1 to G-113; M-1 to R-112; M-1 to G-111; M-1 to R-110; M-1 to L-109; M-1 to D-108; M-1 to C-107; M-1 to P-106; M-1 to S-105; M-1 to G-104; M-1 to 1-103; M-1 to D-102; M-1 to P-100; M-1 to D-100; M-1 to C-99; M-1 to M-98; M-1 to Q-97; M-1 to C-96; M-1 to Q-95; M-1 to L-94; M-1 to C-93; M-1 to K-92; M-1 to S-91; M-1 to L-90; M-1 to G-89; M-1 to N-88; M-1 to L-87; M-1 to H-86; M-1 to A-85; M-1 to I-84; M-1 to Y-83; M-1 to T-82; M-1 to G-81; M-1 to P-80; M-1 to P-79; M-1 to C-78; M-1 to P-77; M-1 to E-76; M-1 to C-75; M-1 to V-74; M-1 to T-73; M-1 to G-72; M-1 to T-71; M-1 to L-70; M-1 to E-69; M-1 to G-68; M-1 to C-67; M-1 to A-66; M-1 to E-65; M-1 to K-64; M-1 to V-63; M-1 to R-62; M-1 to Y-61; M-1 to G-60; M-1 to P-59; M-1 to S-58; M-1 to C-57; M-1 to K-56; M-1 to P-55; M-1 to C-54; M-1 to C-53; M-1 to E-52; M-1 to S-51; M-1 to G-50; M-1 to V-49; M-1 to P-48; M-1 to Y-47; M-1 to E-46; M-1 to D-45; M-1 to E-44; M-1 to K-43; M-1 to C-42; M-1 to S-41; M-1 to P-40; M-1 to L-39; M-1 to A-38; M-1 to P-37; M-1 to A-36; M-1 to Y-35; M-1 to C-34; M-1 to P-33; M-1 to A-32; M-1 to G-31; M-1 to L-30; M-1 to F-29; M-1 to T-28; M-1 to L-27; M-1 to Y-26; M-1 to L-25; M-1 to V-24; M-1 to L-23; M-1 to R-22; M-1 to L-21; M-1 to V-20; M-1 to D-19; M-1 to T-18; M-1 to R-17; M-1 to P-16; M-1 to T-15; M-1 to S-14; M-1 to R-13; M-1 to W-12; M-1 to P-11; M-1 to P-10; M-1 to P-9; M-1 to G-8; M-1 to W-7; and M-1 to D-6 of the sequence of the TR2-SV1 sequence shown in FIG. 4A-4B. The present invention is also directed to nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequences encoding the polypeptides described above. The present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence. Polypeptides encoded by these polynucleotide sequences are also encompassed by the invention.

The invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini of a TR2-SV1 polypeptide, which may be described generally as having residues n2-m2 of FIG. 4A-4B (i.e., SEQ ID NO:5), where n2 and m2 are integers as described above. Polynucleotides encoded by these polypeptides are also encompassed by the invention.

Also mentioned above, even if deletion of one or more amino acids from the N-terminus of a protein results in modification of loss of one or more biological functions of the protein, other biological activities may still be retained. Thus, the ability of shortened TR2-SV2 muteins to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptides generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the N-terminus. Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a TR2-SV2 mutein with a large number of deleted N-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six TR2-SV2 amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the TR2-SV2 amino acid sequence shown in FIG. 7A-7B (i.e., SEQ ID NO:8), up to the serine residue at position number 131 and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues n3-136 of FIG. 7A-7B (i.e., SEQ ID NO:8), where n3 is an integer in the range of 2 to 131. Polynucleotides encoded by these polypeptides are also encompassed by the invention.

More in particular, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues of L-2 to K-136; G-3 to K-136; T-4 to K-136; S-5 to K-136; G-6 to K-136; H-7 to K-136; L-8 to K-136; V-9 to K-136; W-10 to K-136; L-11 to K-136; S-12 to K-136; Q-13 to K-136; G-14 to K-136; F-15 to K-136; S-16 to K-136; L-17 to K-136; A-18 to K-136; G-19 to K-136; R-20 to K-136; P-21 to K-136; G-22 to K-136; S-23 to K-136; S-24 to K-136; P-25 to K-136; W-26 to K-136; P-27 to K-136; V-28 to K-136; D-29 to K-136; A-30 to K-136; V-31 to K-136; L-32 to K-136; A-33 to K-136; C-34 to K-136; G-35 to K-136; W-36 to K-136; C-37 to K-136; P-38 to K-136; G-39 to K-136; L-40 to K-136; H-41 to K-136; V-42 to K-136; P-43 to K-136; P-44 to K-136; L-45 to K-136; S-46 to K-136; P-47 to K-136; S-48 to K-136; S-49 to K-136; W-50 to K-136; T-51 to K-136; P-52 to K-136; A-53 to K-136; M-54 to K-136; G-55 to K-136; L-56 to K-136; R-57 to K-136; A-58 to K-136; S-59 to K-136; R-60 to K-136; N-61 to K-136. C-62 to K-136; S-63 to K-136; R-64 to K-136; T-65 to K-136; E-66 to K-136; N-67 to K-136; A-68 to K-136; V-69 to K-136; C-70 to K-136; G-71 to K-136; C-72 to K-136; S-73 to K-136; P-74 to K-136; G-75 to K-136; H-76 to K-136; F-77 to K-136; C-78 to K-136; I-79 to K-136; V-80 to K-136; Q-81 to K-136; D-82 to K-136; G-83 to K-136; D-84 to K-136; H-85 to K-136; C-86 to K-136; A-87 to K-136; A-88 to K-136; C-89 to K-136; R-90 to K-136; A-91 to K-136; Y-92 to K-136; A-93 to K-136; T-94 to K-136; S-95 to K-136; S-96 to K-136; P-97 to K-136; G-98 to K-136; Q-99 to K-136; R-100 to K-136; V-101 to K-136; Q-102 to K-136; K-103 to K-136; G-104 to K-136; G-105 to K-136; T-106 to K-136; E-107 to K-136; S-108 to K-136; Q-109 to K-136; D-91 to K-136; T-111 to K-136; L-932 to K-136; C-113 to K-136; Q-109 to K-136; N-115 to K-136; C-116 to K-136; P-117 to K-136; R-118 to K-136; G-119 to K-136; P-120 to K-136; S-121 to K-136; L-122 to K-136; P-123 to K-136; M-124 to K-136; G-125 to K-136; P-126 to K-136; W-127 to K-136; R-128 to K-136; N-129 to K-136; V-130 to K-136; and S-131 to K-136 of the TR2-SV2 sequence shown in FIG. 7A-7B. The present invention is also directed to nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequences encoding the polypeptides described above. The present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence. Polypeptides encoded by these polynucleotide sequences are also encompassed by the invention.

Also as mentioned above, even if deletion of one or more amino acids from the C-terminus of a protein results in modification of loss of one or more biological functions of the protein, other biological activities may still be retained. Thus, the ability of the shortened TR2-SV2 mutein to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a TR2-SV2 mutein with a large number of deleted C-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six TR2-SV2 amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the TR2-SV2 polypeptide shown in FIG. 7A-7B (i.e., SEQ ID NO:8), up to the glycine residue at position number 6, and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues 1-m3 of FIG. 7A-7B (i.e., SEQ ID NO:8), where m3 is an integer in the range of 6 to 135. Polynucleotides encoded by these polypeptides are also encompassed by the invention.

More in particular, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues M-1 to S-135; M-1 to P-134; M-1 to R-133; M-1 to T-132; M-1 to S-131; M-1 to V-130; M-1 to N-129; M-1 to R-128; M-1 to W-127; M-1 to P-126; M-1 to G-125; M-1 to M-124; M-1 to P-123; M-1 to L-122; M-1 to S-121; M-1 to P-120; M-1 to G-119; M-1 to R-118; M-1 to P-117; M-1 to C-116; M-1 to N-115; M-1 to Q-114; M-1 to C-113; M-1 to L-112; M-1 to T-111; M-1 to D-110; M-1 to Q-109; M-1 to S-108; M-1 to E-107; M-1 to T-106; M-1 to G-105; M-1 to G-104; M-1 to K-103; M-1 to Q-102; M-1 to V-101; M-1 to R-100; M-1 to Q-99; M-1 to G-98; M-1 to P-97; M-1 to S-96; M-1 to S-95; M-1 to T-94; M-1 to A-93; M-1 to Y-92; M-1 to A-91; M-1 to R-90; M-1 to C-89; M-1 to A-88; M-1 to A-87; M-1 to C-86; M-1 to H-85; M-1 to D-84; M-1 to G-83; M-1 to D-82; M-1 to Q-81; M-1 to V-80; M-1 to 1-79; M-1 to C-78; M-1 to F-77; M-1 to H-76; M-1 to G-75; M-1 to P-74; M-1 to S-73; M-1 to C-72; M-1 to G-71; M-1 to C-70; M-1 to V-69; M-1 to A-68; M-1 to N-67; M-1 to E-66; M-1 to T-65; M-1 to R-64; M-1 to S-63; M-1 to C-62; M-1 to N-61; M-1 to R-60; M-1 to S-59; M-1 to A-58; M-1 to R-57; M-1 to L-56; M-1 to G-55; M-1 to M-54; M-1 to A-53; M-1 to P-52; M-1 to T-51; M-1 to W-50; M-1 to S-49; M-1 to S-48; M-1 to P-47; M-1 to S-46; M-1 to L-45; M-1 to P-44; M-1 to P-43; M-1 to V-42; M-1 to H-41; M-1 to L-40; M-1 to G-39; M-1 to P-38; M-1 to C-37; M-1 to W-36; M-1 to G-35; M-1 to C-34; M-1 to A-33; M-1 to L-32; M-1 to V-31; M-1 to A-30; M-1 to D-29; M-1 to V-28; M-1 to P-27; M-1 to W-26; M-1 to P-25; M-1 to S-24; M-1 to S-23; M-1 to G-22; M-1 to P-21; M-1 to R-20; M-1 to G-19; M-1 to A-18; M-1 to L-17; M-1 to S-16; M-1 to F-15; M-1 to G-14; M-1 to Q-13; M-1 to S-12; M-1 to L-11; M-1 to W-10; M-1 to V-9; M-1 to L-8; M-1 to H-7; and M-1 to G-6 of the sequence of the TR2-SV2 sequence shown in FIG. 7A-7B. The present invention is also directed to nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequences encoding the polypeptides described above. The present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence. Polypeptides encoded by these polynucleotide sequences are also encompassed by the invention.

The invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini of a TR2-SV2 polypeptide, which may be described generally as having residues n3-m3 of FIG. 7A-7B (i.e., SEQ ID NO:8), where n3 and m3 are integers as described above. Polynucleotides encoded by these polypeptides are also encompassed by the invention.

In addition, the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the predicted extracellular domain of the TR2 amino acid sequence shown in SEQ ID NO:2 (FIG. 1A-1B), up to the glycine residue at position number 159 and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues n4-164 of SEQ ID NO:2, where n4 is an integer in the range of 1 to 159. Polynucleotides encoded by these polypeptides are also encompassed by the invention.

More in particular, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues of P-1 to H-164; A-2 to H-164; L-3 to H-164; P-4 to H-164; S-5 to H-164; C-6 to H-164; K-7 to H-164; E-8 to H-164; D-9 to H-164; E-10 to H-164; Y-11 to H-164; P-12 to H-164; V-13 to H-164; G-14 to H-164; S-15 to H-164; E-16 to H-164; C-17 to H-164; C-18 to H-164; P-19 to H-164; K-20 to H-164; C-21 to H-164; S-22 to H-164; P-23 to H-164; G-24 to H-164; Y-25 to H-164; R-26 to H-164; V-27 to H-164; K-28 to H-164; E-29 to H-164; A-30 to H-164; C-31 to H-164; G-32 to H-164; E-33 to H-164; L-34 to H-164; T-35 to H-164; G-36 to H-164; T-37 to H-164; V-38 to H-164; C-39 to H-164; E-40 to H-164; P-41 to H-164; C-42 to H-164; P-43 to H-164; P-44 to H-164; G-45 to H-164; T-46 to H-164; Y-47 to H-164; I-48 to H-164; A-49 to H-164; H-50 to H-164; L-51 to H-164; N-52 to H-164; G-53 to H-164; L-54 to H-164; S-55 to H-164; K-56 to H-164; C-57 to H-164; L-58 to H-164; Q-59 to H-164; C-60 to H-164; Q-61 to H-164; M-62 to H-164; C-63 to H-164; D-64 to H-164; P-65 to H-164; A-66 to H-164; M-67 to H-164; G-68 to H-164; L-69 to H-164; R-70 to H-164; A-71 to H-164; S-72 to H-164; R-73 to H-164; N-74 to H-164; C-75 to H-164; S-76 to H-164; R-77 to H-164; T-78 to H-164; E-79 to H-164; N-80 to H-164; A-81 to H-164; V-82 to H-164; C-83 to H-164; G-84 to H-164; C-85 to H-164; S-86 to H-164; P-87 to H-164; G-88 to H-164; H-89 to H-164; F-90 to H-164; C-91 to H-164; I-92 to H-164; V-93 to H-164; Q-94 to H-164; D-95 to H-164; G-96 to H-164; D-97 to H-164; H-98 to H-164; C-99 to H-164; A-100 to H-164; A-101 to H-164; C-102 to H-164; R-103 to H-164; A-104 to H-164; Y-105 to H-164; A-106 to H-164; T-107 to H-164; S-108 to H-164; S-109 to H-164; P-110; to H-164; G-111 to H-164; Q-112 to H-164; R-113 to H-164; V-114 to H-164; Q-115 to H-164; K-116 to H-164; G-117 to H-164; G-118 to H-164; T-119 to H-164; E-120 to H-164; S-121 to H-164; Q-122 to H-164; D-123 to H-164; T-124 to H-164; L-125 to H-164; C-126 to H-164; Q-127 to H-164; N-128 to H-164; C-129 to H-164; P-130 to H-164; P-131 to H-164; G-132 to H-164; T-133 to H-164; F-134 to H-164; S-135 to H-164; P-136 to H-164; N-137 to H-164; G-138 to H-164; T-139 to H-164; L-140 to H-164; E-141 to H-164; E-142 to H-164; C-143 to H-164; Q-144 to H-164; H-145 to H-164; Q-146 to H-164; T-147 to H-164; K-148 to H-164; C-149 to H-164; S-150 to H-164; W-151 to H-164; L-152 to H-164; V-153 to H-164; T-154 to H-164; K-155 to H-164; A-156 to H-164; G-157 to H-164; A-158 to H-164; and G-159 to H-164 of the TR2 amino acid sequence shown in SEQ ID NO:2 (which is identical to that shown in FIG. 1A-1B, with the exception that the amino acid residues in FIG. 1A-1B are numbered consecutively from 1 through 283 from the N-terminus to the C-terminus, while the amino acid residues in SEQ ID NO:2 are numbered consecutively from −36 through 247 to reflect the position of the predicted signal peptide). The present invention is also directed to nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequences encoding the polypeptides described above. The present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence. Polypeptides encoded by these polynucleotide sequences are also encompassed by the invention.

The present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the predicted extracellular domain of the amino acid sequence of the TR2 shown in SEQ ID NO:2 (FIG. 1A-1B), up to the cysteine residue at position number 6 in SEQ ID NO:2, and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues 1-m4 of SEQ ID NO:2 (FIG. 1A-1B), where m4 is an integer in the range of 6 to 164. Polynucleotides encoded by these polypeptides are also encompassed by the invention.

More in particular, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues P-1 to H-164; P-1 to S-163; P-1 to S-162; P-1 to S-161; P-1 to T-160; P-1 to G-159; P-1 to A-158; P-1 to G-157; P-1 to A-156; P-1 to K-155; P-1 to T-154; P-1 to V-153; P-1 to L-152; P-1 to W-151; P-1 to S-150; P-1 to C-149; P-1 to K-148; P-1 to T-147; P-1 to Q-146; P-1 to H-145; P-1 to Q-144; P-1 to C-143; P-1 to E-142; P-1 to E-141; P-1 to L-140; P-1 to T-139; P-1 to G-138; P-1 to N-137; P-1 to P-136; P-1 to S-135; P-1 to F-134; P-1 to T-133; P-1 to G-132; P-1 to P-131; P-1 to P-130; P-1 to C-129; P-1 to N-128; P-1 to Q-127; P-1 to C-126; P-1 to L-125; P-1 to T-124; P-1 to D-123; P-1 to Q-122; P-1 to S-121; P-1 to E-120; P-1 to T-119; P-1 to G-118; P-1 to G-117; P-1 to K-116; P-1 to Q-115; P-1 to V-114; P-1 to R-113; P-1 to Q-112; P-1 to G-111; P-1 to P-110; P-1 to S-109; P-1 to S-108; P-1 to T-107; P-1 to A-106; P-1 to Y-105; P-1 to A-104; P-1 to R-103; P-1 to C-102; P-1 to A-101; P-1 to A-100; P-1 to C-99; P-1 to H-98; P-1 to D-97; P-1 to G-96; P-1 to D-95; P-1 to Q-94; P-1 to V-93; P-1 to 1-92; P-1 to C-91; P-1 to F-90; P-1 to H-89; P-1 to G-88; P-1 to P-87; P-1 to S-86; P-1 to C-85; P-1 to G-84; P-1 to C-83; P-1 to V-82; P-1 to A-81; P-1 to N-80; P-1 to E-79; P-1 to T-78; P-1 to R-77; P-1 to S-76; P-1 to C-75; P-1 to N-74; P-1 to R-73; P-1 to S-72; P-1 to A-71; P-1 to R-70; P-1 to L-69; P-1 to G-68; P-1 to M-67; P-1 to A-66; P-1 to P-65; P-1 to D-64; P-1 to C-63; P-1 to M-62; P-1 to Q-61; P-1 to C-60; P-1 to Q-59; P-1 to L-58; P-1 to C-57; P-1 to K-56; P-1 to S-55; P-1 to L-54; P-1 to G-53; P-1 to N-52; P-1 to L-51; P-1 to H-50; P-1 to A-49; P-1 to 1-48; P-1 to Y-47; P-1 to T-46; P-1 to G-45; P-1 to P-44; P-1 to P-43; P-1 to C-42; P-1 to P-41; P-1 to E-40; P-1 to C-39; P-1 to V-38; P-1 to T-37; P-1 to G-36; P-1 to T-35; P-1 to L-34; P-1 to E-33; P-1 to G-32; P-1 to C-31; P-1 to A-30; P-1 to E-29; P-1 to K-28; P-1 to V-27; P-1 to R-26; P-1 to Y-25; P-1 to G-24; P-1 to P-23; P-1 to S-22; P-1 to C-21; P-1 to K-20; P-1 to P-19; P-1 to C-18; P-1 to C-17; P-1 to E-16; P-1 to S-15; P-1 to G-14; P-1 to V-13; P-1 to P-12; P-1 to Y-11; P-1 to E-10; P-1 to D-9; P-1 to E-8; P-1 to K-7; and P-1 to C-6 of the sequence of the TR2 sequence shown in SEQ ID NO:2 (which is identical to the sequence shown as FIG. 1A-1B, with the exception that the amino acid residues in FIG. 1A-1B are numbered consecutively from 1 through 283 from the N-terminus to the C-terminus, while the amino acid residues in SEQ ID NO:2 are numbered consecutively from −36 through 247 to reflect the position of the predicted signal peptide). The present invention is also directed to nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequences encoding the polypeptides described above. The present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence. Polypeptides encoded by these polynucleotide sequences are also encompassed by the invention.

The invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini of a soluble TR2 polypeptide, which may be described generally as having residues n4-m4 of SEQ ID NO:2 (FIG. 1A-1B), where n4 and m4 are integers as described above. Polynucleotides encoded by these polypeptides are also encompassed by the invention.

In addition, the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the predicted extracellular domain of the TR2-SV1 amino acid sequence shown in SEQ ID NO:5 (FIG. 4A-4B), up to the threonine residue at position number 144 and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues n5-149 of SEQ ID NO:5, where n5 is an integer in the range of 1 to 144. Polynucleotides encoded by these polypeptides are also encompassed by the invention.

More in particular, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues of P-1 to A-149; A-2 to A-149; L-3 to A-149; P-4 to A-149; S-5 to A-149; C-6 to A-149; K-7 to A-149; E-8 to A-149; D-9 to A-149; E-10 to A-149; Y-11 to A-149; P-12 to A-149; V-13 to A-149; G-14 to A-149; S-15 to A-149; E-16 to A-149; C-17 to A-149; C-18 to A-149; P-19 to A-149; K-20 to A-149; C-21 to A-149; S-22 to A-149; P-23 to A-149; G-24 to A-149; Y-25 to A-149; R-26 to A-149; V-27 to A-149; K-28 to A-149; E-29 to A-149; A-30 to A-149; C-31 to A-149; G-32 to A-149; E-33 to A-149; L-34 to A-149; T-35 to A-149; G-36 to A-149; T-37 to A-149; V-38 to A-149; C-39 to A-149; E-40 to A-149; P-41 to A-149; C-42 to A-149; P-43 to A-149; P-44 to A-149; G-45 to A-149; T-46 to A-149; Y-47 to A-149; 1-48 to A-149; A-49 to A-149; H-50 to A-149; L-51 to A-149; N-52 to A-149; G-53 to A-149; L-54 to A-149; S-55 to A-149; K-56 to A-149; C-57 to A-149; L-58 to A-149; Q-59 to A-149; C-60 to A-149; Q-61 to A-149; M-62 to A-149; C-63 to A-149; D-64 to A-149; P-65 to A-149; D-66 to A-149; 1-67 to A-149; G-68 to A-149; S-69 to A-149; P-70 to A-149; C-71 to A-149; D-72 to A-149; L-73 to A-149; R-74 to A-149; G-75 to A-149; R-76 to A-149; G-77 to A-149; H-78 to A-149; L-79 to A-149; E-80 to A-149; A-81 to A-149; G-82 to A-149; A-83 to A-149; H-84 to A-149; L-85 to A-149; S-86 to A-149; P-87 to A-149; G-88 to A-149; R-89 to A-149; Q-90 to A-149; K-91 to A-149; G-92 to A-149; E-93 to A-149; P-94 to A-149; D-95 to A-149; P-96 to A-149; E-97 to A-149; V-98 to A-149; A-99 to A-149; F-100 to A-149; E-101 to A-149; S-102 to A-149; L-103 to A-149; S-104 to A-149; A-105 to A-149; E-106 to A-149; P-107 to A-149; V-108 to A-149; H-109 to A-149; A-110 to A-149; A-111 to A-149, N-112 to A-149; G-113 to A-149; S-114 to A-149; V-115 to A-149; P-116 to A-149; L-117 to A-149; E-118 to A-149; P-119 to A-149; H-120 to A-149; A-121 to A-49; R-122 to A-149; L-123 to A-149; S-124 to A-149; M-125 to A-149; A-126 to A-149; S-127 to A-149; A-128 to A-149; P-129 to A-149; C-130 to A-149; G-131 to A-149; Q-132 to A-149; A-133 to A-149; G-134 to A-149; L-135 to A-149; H-136 to A-149; L-137 to A-149; R-138 to A-149; D-139 to A-149; R-140 to A-149; A-141 to A-149; D-142 to A-149; G-143 to A-149; and T-144 to A-149 of the TR2-SV1 amino acid sequence shown in SEQ ID NO:5 (which is identical to that shown in FIG. 4A-4B, with the exception that the amino acid residues in FIG. 4A-4B are numbered consecutively from 1 through 185 from the N-terminus to the C-terminus, while the amino acid residues in SEQ ID NO:5 are numbered consecutively from −36 through 149 to reflect the position of the predicted signal peptide). The present invention is also directed to nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequences encoding the polypeptides described above. The present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence. Polypeptides encoded by these polynucleotide sequences are also encompassed by the invention.

The present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the predicted extracellular domain of the amino acid sequence of the TR2-SV1 shown in SEQ ID NO:5 (FIG. 4A-4B), up to the cysteine residue at position number 6 in SEQ ID NO:5, and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues 1-m5 of SEQ ID NO:5 (FIG. 4A-4B), where m5 is an integer in the range of 6 to 149. Polynucleotides encoded by these polypeptides are also encompassed by the invention.

More in particular, the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues P-1 to A-149; P-1 to R-148; P-1 to G-147; P-1 to G-146; P-1 to P-145; P-1 to T-144; P-1 to G-143; P-1 to D-142; P-1 to A-141; P-1 to R-140; P-1 to D-139; P-1 to R-138; P-1 to L-137; P-1 to H-136; P-1 to L-135; P-1 to G-134; P-1 to A-133; P-1 to Q-132; P-1 to G-131; P-1 to C-130; P-1 to P-129; P-1 to A-128; P-1 to S-127; P-1 to A-126; P-1 to M-125; P-1 to S-124; P-1 to L-123; P-1 to R-122; P-1 to A-121; P-1 to H-120; P-1 to P-119; P-1 to E-118; P-1 to L-117; P-1 to P-116; P-1 to V-115; P-1 to S-114; P-1 to G-113; P-1 to N-112; P-1 to A-111; P-1 to A-110; P-1 to H-109; P-1 to V-108; P-1 to P-107; P-1 to E-106; P-1 to A-105; P-1 to S-104; P-1 to L-103; P-1 to S-102; P-1 to E-101; P-1 to F-100; P-1 to A-99; P-1 to V-98; P-1 to E-97; P-1 to P-96; P-1 to D-95; P-1 to P-94; P-1 to E-93; P-1 to G-92; P-1 to K-91; P-1 to Q-90; P-1 to R-89; P-1 to G-88; P-1 to P-87; P-1 to S-86; P-1 to L-85; P-1 to H-84; P-1 to A-83; P-1 to G-82; P-1 to A-81; P-1 to E-80; P-1 to L-79; P-1 to H-78; P-1 to G-77; P-1 to R-76; P-1 to G-75; P-1 to R-74; P-1 to L-73; P-1 to D-72; P-1 to C-71; P-1 to P-70; P-1 to S-69; P-1 to G-68; P-1 to 1-67; P-1 to D-66; P-1 to P-65; P-1 to D-64; P-1 to C-63; P-1 to M-62; P-1 to Q-61; P-1 to C-60; P-1 to Q-59; P-1 to L-58; P-1 to C-57; P-1 to K-56; P-1 to S-55; P-1 to L-54; P-1 to G-53; P-1 to N-52; P-1 to L-51; P-1 to H-50; P-1 to A-49; P-1 to 1-48; P-1 to Y-47; P-1 to T-46; P-1 to G-45; P-1 to P-44; P-1 to P-43; P-1 to C-42; P-1 to P-41; P-1 to E-40; P-1 to C-39; P-1 to V-38; P-1 to T-37; P-1 to G-36; P-1 to T-35; P-1 to L-34; P-1 to E-33; P-1 to G-32; P-1 to C-31; P-1 to A-30; P-1 to E-29; P-1 to K-28; P-1 to V-27; P-1 to R-26; P-1 to Y-25; P-1 to G-24; P-1 to P-23; P-1 to S-22; P-1 to C-21; P-1 to K-20; P-1 to P-19; P-1 to C-18; P-1 to C-17; P-1 to E-16; P-1 to S-15; P-1 to G-14; P-1 to V-13; P-1 to P-12; P-1 to Y-11; P-1 to E-10; P-1 to D-9; P-1 to E-8; P-1 to K-7; and P-1 to C-6 of the sequence of the TR2-SV1 sequence shown in SEQ ID NO:5 (which is identical to the sequence shown as FIG. 4A-4B, with the exception that the amino acid residues in FIG. 4A-4B are numbered consecutively from 1 through 185 from the N-terminus to the C-terminus, while the amino acid residues in SEQ ID NO:5 are numbered consecutively from −36 through 149 to reflect the position of the predicted signal peptide). The present invention is also directed to nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequences encoding the polypeptides described above. The present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence. Polypeptides encoded by these polynucleotide sequences are also encompassed by the invention.

The invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini of a soluble TR2-SV1 polypeptide, which may be described generally as having residues n5-m5 of SEQ ID NO:5 (FIG. 4A-4B), where n5 and m5 are integers as described above. Polynucleotides encoded by these polypeptides are also encompassed by the invention.

Additionally, one or more of the amino acid residues of the polypeptides of the invention (e.g., arginine and lysine residues) may be deleted or substituted with another residue to eliminate undesired processing by proteases such as, for example, furins or kexins.

The invention further provides for the proteins containing polypeptide sequences encoded by the polynucleotides of the invention.

Among the especially preferred fragments of the invention are fragments characterized by structural or functional attributes of TR2 receptors of the invention. Such fragments include amino acid residues that comprise, or alternatively consist of, alpha-helix and alpha-helix forming regions (“alpha-regions”), beta-sheet and beta-sheet-forming regions (“beta-regions”), turn and turn-forming regions (“turn-regions”), coil and coil-forming regions (“coil-regions”), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, surface forming regions, and high antigenic index regions (i.e., containing four or more contiguous amino acids having an antigenic index of greater than or equal to 1.5, as identified using the default parameters of the Jameson-Wolfprogram) of complete (i.e., full-length) TR2 receptor (SEQ ID NO:2). Certain preferred regions are those set out in FIG. 3 and include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence depicted in FIG. 1A-1B (SEQ ID NO:2), such preferred regions include; Garnier-Robson predicted alpha-regions, beta-regions, turn-regions, and coil-regions; Chou-Fasman predicted alpha-regions, beta-regions, turn-regions, and coil-regions; Kyte-Doolittle predicted hydrophilic and hydrophobic regions; Eisenberg alpha and beta amphipathic regions; Emini surface-forming regions; and Jameson-Wolf high antigenic index regions, as predicted using the default parameters of these computer programs. Polynucleotides encoding these polypeptides are also encompassed by the invention.

In additional embodiments, the polynucleotides of the invention encode functional attributes of TR2 receptors. Preferred embodiments of the invention in this regard include fragments that comprise, or alternatively consist of, one, two, three, four or more of one or more of the following functional domains: alpha-helix and alpha-helix forming regions (“alpha-regions”), beta-sheet and beta-sheet forming regions (“beta-regions”), turn and turn-forming regions (“turn-regions”), coil and coil-forming regions (“coil-regions”), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions of TR2 receptors.

The data representing the structural or functional attributes of the TR2 receptors set forth in FIG. 3, 6 and 9 and Tables II, II and IX were generated using the various identified modules and algorithms of the DNA*STAR set on default parameters. In a preferred embodiment, the data presented in columns VIII, IX, XIII, and XIV of Table II can be used to determine regions of TR2 receptors which exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII, IX, XIII, and/or IV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.

Certain preferred regions in these regards are set out in FIG. 3, 6 and 9, but may, as shown in Tables II, III and IV, respectively, be represented or identified by using tabular representations of the data presented in FIG. 3, 6 and 9. The DNA*STAR computer algorithm used to generate FIG. 3, 6 and 9 (set on the original default parameters) was used to present the data in FIG. 3, 6 and 9 in a tabular format. (See Tables II, III and IV, respectively).

The above-mentioned preferred regions set out in FIG. 3, 6 and 9 and in Tables II, III and IV include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence set out in FIG. 1, 4 and 7. As set out in FIG. 3, 6 and 9, and in Tables II, III and IV, such preferred regions include Garnier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions (columns I, III, V, and VII in Tables II, III and IV), Chou-Fasman alpha-regions, beta-regions, and turn-regions (columns II, IV, and VI in Tables II, III and IV), Kyte-Doolittle hydrophilic regions (column VIII in Tables II, III and IV), Hopp-Woods hydrophobic regions (column IX in Tables II, III and IV), Eisenberg alpha- and beta-amphipathic regions (columns X and XI in Tables II, III and IV), Karplus-Schulz flexible regions (column XII in Tables II, III and IV), Jameson-Wolf regions of high antigenic index (column XIII in Tables II, III and IV), and Emini surface-forming regions (column XIV in Tables II, III and IV).

TABLE II
Res Position I II III IV V VI VII VIII IX X XI XII XIII XIV
Met 1 . . B . . . . 0.58 −0.24 . . . 1.15 1.53
Glu 2 . . B . . . . 0.97 −0.24 . * . 1.40 1.19
Pro 3 . . . . . T C 1.07 −0.67 * * . 2.35 1.55
Pro 4 . . . . T T . 1.11 −0.19 * * . 2.50 1.65
Gly 5 . . . . T T . 1.29 −0.37 . * F 2.25 0.94
Asp 6 . . . . T T . 1.68 0.06 * * F 1.40 0.94
Trp 7 . . . . T . . 1.47 0.06 . . F 0.95 0.94
Gly 8 . . . . . . C 1.39 0.06 * * F 0.65 1.47
Pro 9 . . . . . T C 1.71 0.54 * * F 0.15 0.93
Pro 10 . . . . . T C 1.76 0.54 * * F 0.30 1.73
Pro 11 . . . . T T . 1.44 0.01 * * F 0.80 2.34
Trp 12 . . . . T T . 1.52 0.07 * * F 0.80 2.18
Arg 13 . . . . T . . 1.91 0.07 * * F 0.94 2.18
Ser 14 . . . . . . C 1.81 −0.36 * * F 1.68 2.82
Thr 15 . . . . . T C 2.02 −0.30 * * F 2.22 3.88
Pro 16 . . . . . T C 1.38 −1.21 . * F 2.86 3.30
Lys 17 . . . . T T . 0.86 −0.57 . * F 3.40 1.83
Thr 18 . . B . . T . 0.86 −0.27 . * F 2.36 1.05
Asp 19 . . B B . . . 0.34 −0.76 * . F 1.92 1.32
Val 20 . . B B . . . −0.20 −0.50 * * . 1.28 0.55
Leu 21 . . B B . . . −0.80 0.14 * . . 0.04 0.28
Arg 22 . . B B . . . −1.09 0.34 * * . −0.30 0.14
Leu 23 . . B B . . . −1.59 1.10 * * . −0.60 0.29
Val 24 . . B B . . . −1.90 1.14 * * . −0.60 0.29
Leu 25 . . B B . . . −1.74 0.94 * * . −0.60 0.22
Tyr 26 . . B B . . . −1.74 1.73 * * . −0.60 0.23
Leu 27 . . B B . . . −2.20 1.73 * * . −0.60 0.25
Thr 28 . . B B . . . −1.98 1.51 . . . −0.60 0.30
Phe 29 . . B B . . . −1.33 1.33 . . . −0.60 0.19
Leu 30 . . B B . . . −1.19 1.00 . . . −0.60 0.37
Gly 31 . . B B . . . −1.19 0.89 . . . −0.60 0.14
Ala 32 . . B . . T . −0.97 1.16 . . . −0.20 0.25
Pro 33 . . . . T T . −0.87 0.87 . . . 0.20 0.30
Cys 34 . . . . T T . −0.76 0.61 . . . 0.20 0.47
Tyr 35 . . B . . T . −0.76 0.69 . . . −0.20 0.47
Ala 36 . . B . . . . −0.62 0.87 . . . −0.40 0.25
Pro 37 . . B . . . . −0.33 0.87 . . . −0.40 0.72
Ala 38 . . B . . . . −0.79 0.69 . . . −0.14 0.62
Leu 39 . . B . . T . −0.08 0.50 . . . 0.32 0.33
Pro 40 . . B . . T . 0.17 0.00 . . F 1.03 0.42
Ser 41 . . . . T T . 0.76 −0.43 . . F 2.29 0.73
Cys 42 . . B . . T . 0.97 −0.93 . . F 2.60 1.47
Lys 43 . A B . . . . 1.31 −1.61 . . F 1.94 1.65
Glu 44 . A B . . . . 1.91 −1.29 . . F 1.68 1.93
Asp 45 . A . . T . . 1.27 −1.24 . . F 1.82 5.56
Glu 46 . A B . . . . 1.22 −1.17 . . F 1.16 2.06
Tyr 47 . A B . . . . 1.59 −0.74 . . . 0.75 1.18
Pro 48 . . . . T . . 1.54 −0.36 . . . 0.90 0.95
Val 49 . . . . T . . 0.88 −0.36 . . F 1.05 0.95
Gly 50 . . . . T T . 0.21 0.21 . . F 0.65 0.32
Ser 51 . . . . T T . 0.00 0.03 . * F 0.65 0.11
Glu 52 . . . . T T . 0.29 0.03 . * F 0.90 0.23
Cys 53 . . B . . T . −0.17 −0.61 . * . 1.50 0.47
Cys 54 . . B . . T . 0.39 −0.47 . * . 1.45 0.19
Pro 55 . . . . T T . 0.52 −0.47 . . . 2.10 0.15
Lys 56 . . . . T T . 0.48 −0.04 . . F 2.50 0.42
Cys 57 . . . . T T . 0.23 −0.19 * * F 2.25 0.78
Ser 58 . . . . . T C 1.01 0.00 * * F 1.20 0.79
Pro 59 . . . . T T . 0.82 −0.43 . * F 1.75 0.77
Gly 60 . . . . T T . 1.08 0.21 . * F 1.05 1.07
Tyr 61 . . B . . T . 1.03 −0.36 . * . 0.85 1.60
Arg 62 . A B . . . . 1.11 −0.74 * * . 0.75 1.79
Val 63 . A B . . . . 0.74 −0.67 * * . 0.75 1.83
Lys 64 . A B . . . . 0.61 −0.53 * * . 0.60 0.63
Glu 65 . A B . . . . 0.96 −0.86 * * . 0.60 0.32
Ala 66 . A B . . . . 0.39 −0.86 * * . 0.60 0.74
Cys 67 . A B . . . . −0.03 −0.81 * * . 0.60 0.30
Gly 68 . A . . T . . 0.48 −0.33 * * . 0.70 0.25
Glu 69 . A . . T . . 0.12 0.10 * * F 0.25 0.25
Leu 70 . . . B T . . −0.73 0.09 * * F 0.25 0.67
Thr 71 . . . B T . . −0.81 0.16 * * F 0.25 0.50
Gly 72 . . . B T . . −0.14 0.30 * * F 0.25 0.16
Thr 73 . . . B T . . −0.01 0.30 * * F 0.25 0.33
Val 74 . . B B . . . −0.68 0.04 * * . −0.30 0.35
Cys 75 . . B B . . . −0.08 0.13 . . . −0.30 0.19
Glu 76 . . B B . . . 0.02 0.13 . . . −0.23 0.20
Pro 77 . . . . T . . 0.02 0.07 . . F 0.59 0.42
Cys 78 . . . . . . C 0.02 −0.14 . . F 1.06 0.78
Pro 79 . . . . . T C 0.63 −0.23 . . F 1.33 0.65
Pro 80 . . . . T T . 0.41 0.53 . . F 0.70 0.66
Gly 81 . . . . T T . −0.18 0.79 . . F 0.63 0.86
Thr 82 . . B . . T . 0.00 0.71 . . F 0.16 0.56
Tyr 83 . . B . . . . −0.14 0.79 . . . −0.26 0.49
Ile 84 . . B . . . . 0.07 1.04 . . . −0.33 0.41
Ala 85 . . B . . . . −0.07 1.01 . . . −0.40 0.46
His 86 . . B . . T . −0.53 0.96 . . . −0.20 0.29
Leu 87 . . B . . T . −0.52 0.89 * . . −0.20 0.34
Asn 88 . . . . T T . −0.23 0.59 * * . 0.20 0.45
Gly 89 . . . . T T . −0.01 0.09 * . F 0.65 0.66
Leu 90 . . . . T . . −0.23 0.16 * * F 0.45 0.43
Ser 91 . . . . T T . −0.20 0.16 * . F 0.65 0.22
Lys 92 . . . . T T . −0.06 0.16 * * . 0.50 0.39
Cys 93 . . B . . T . −0.06 0.30 * * . 0.10 0.25
Leu 94 . . B . . T . −0.31 0.01 * . . 0.10 0.33
Gln 95 . A B . . . . −0.17 0.24 * . . −0.30 0.16
Cys 96 . A B . . . . 0.13 0.81 * . . −0.60 0.16
Gln 97 . A B . . . . −0.12 0.24 * . . −0.30 0.33
Met 98 . A B . . . . −0.04 −0.01 . . . 0.30 0.29
Cys 99 . A B . . . . 0.17 0.09 . * . −0.30 0.55
Asp 100 . . B . . . . −0.18 0.13 . . . −0.10 0.31
Pro 101 . . B . . . . −0.32 0.16 . * . −0.10 0.31
Ala 102 . A B . . . . −0.21 0.23 . * . −0.30 0.48
Met 103 . A B . . . . −0.20 −0.34 . * . 0.30 0.57
Gly 104 . A B . . . . 0.17 0.16 * * . −0.30 0.37
Leu 105 . A B . . . . 0.28 0.11 * * . −0.30 0.49
Arg 106 . A B . . . . 0.49 −0.39 * * . 0.64 0.97
Ala 107 . A B . . . . 0.41 −0.60 * * . 1.43 1.58
Ser 108 . . . . T T . 0.71 −0.46 * * F 2.42 1.03
Arg 109 . . . . T T . 1.17 −0.76 * * F 2.91 0.70
Asn 110 . . . . T T . 1.67 −0.76 * * F 3.40 1.36
Cys 111 . . . . T T . 1.56 −0.77 * * F 3.06 1.47
Ser 112 . . . . T . . 2.14 −1.16 * . F 2.52 1.30
Arg 113 . A . . T . . 1.86 −0.76 * * F 1.98 1.30
Thr 114 . A . . T . . 0.89 −0.66 * * F 1.64 2.44
Glu 115 . A . . T . . 0.22 −0.59 * . F 1.30 1.35
Asn 116 . A B . . . . 0.54 −0.40 * . F 0.45 0.37
Ala 117 . A B . . . . 0.18 0.03 * . . −0.30 0.25
Val 118 . A . . T . . −0.23 0.11 * * . 0.10 0.08
Cys 119 . . . . T . . −0.13 0.50 . . . 0.00 0.07
Gly 120 . . . . T . . −0.48 0.53 . . . 0.00 0.10
Cys 121 . . . . T . . −0.51 0.46 . . . 0.00 0.13
Ser 122 . . . . . T C −0.62 0.31 . . . 0.30 0.34
Pro 123 . . . . T T . −0.43 0.53 . * F 0.35 0.30
Gly 124 . . . . T T . −0.66 0.67 . . . 0.20 0.30
His 125 . . B . . T . −1.17 0.79 . . . −0.20 0.16
Phe 126 . . B B . . . −0.50 1.04 . . . −0.60 0.07
Cys 127 . . B B . . . −0.20 1.01 . . . −0.32 0.13
Ile 128 . . B B . . . −0.33 0.59 . * . −0.04 0.16
Val 129 . . B . . T . 0.01 0.51 . * . 0.64 0.18
Gln 130 . . . . T T . 0.01 −0.27 . . F 2.37 0.57
Asp 131 . . . . T T . 0.04 −0.34 * . F 2.80 1.11
Gly 132 . . . . T T . 0.12 −0.46 * . F 2.37 0.80
Asp 133 . A . . T . . 0.42 −0.60 * . F 1.99 0.47
His 134 . A . . T . . 0.61 −0.50 . * . 1.56 0.28
Cys 135 . A B . . . . 0.72 0.07 . * . −0.02 0.15
Ala 136 . A B . . . . 0.13 −0.36 . * . 0.30 0.18
Ala 137 . A B . . . . 0.23 0.14 . * . −0.30 0.13
Cys 138 . A B . . . . −0.36 0.40 . * . −0.30 0.39
Arg 139 . A B . . . . −0.63 0.33 . * . −0.30 0.39
Ala 140 . A B . . . . −0.27 0.31 . * . −0.30 0.56
Tyr 141 . . B . . . . 0.02 0.20 . * . 0.05 1.39
Ala 142 . . B . . . . 0.40 0.01 * * . −0.10 0.95
Thr 143 . . . . T . . 0.72 0.44 . * F 0.30 1.45
Ser 144 . . . . . . C 0.61 0.37 * * F 0.25 0.92
Ser 145 . . . . . T C 1.31 0.01 * * F 0.60 1.57
Pro 146 . . . . . T C 0.70 −0.49 * * F 1.20 2.14
Gly 147 . . . . T T . 1.29 −0.33 * . F 1.40 1.18
Gln 148 . . B . . T . 1.64 −0.31 * . F 1.00 1.53
Arg 149 . . B . . . . 1.60 −0.70 * . F 1.40 1.98
Val 150 . . B . . . . 1.56 −0.70 * . F 1.70 1.98
Gln 151 . . B . . T . 1.46 −0.70 * . F 2.20 1.13
Lys 152 . . B . . T . 1.80 −0.61 * . F 2.35 0.83
Gly 153 . . . . . T C 1.50 −0.61 * . F 3.00 1.94
Gly 154 . . . . . T C 1.39 −0.87 * * F 2.70 1.50
Thr 155 . . . . . . C 2.24 −0.87 * . F 2.45 1.30
Glu 156 . . . . . . C 1.93 −0.87 * . F 2.40 2.20
Ser 157 . . B . T T . 1.08 −0.81 * . F 2.75 3.20
Gln 158 . . . . T T . 0.76 −0.56 * . F 2.70 1.83
Asp 159 . . . . T T . 1.10 −0.47 * . F 2.50 0.57
Thr 160 . . B . . T . 1.41 −0.07 * . F 1.85 0.73
Leu 161 . . . . T . . 0.74 −0.06 * . . 1.65 0.68
Cys 162 . . . . T T . 0.83 0.11 . . . 1.00 0.22
Gln 163 . . . . T T . 0.62 0.54 . . . 0.45 0.23
Asn 164 . . . . T T . 0.28 0.49, * . . 0.20 0.44
Cys 165 . . B . . T . 0.28 0.23 . . F 0.25 0.81
Pro 166 . . . . . T C 0.39 0.14 . . F 0.45 0.67
Pro 167 . . . . T T . 0.76 0.53 . . F 0.35 0.36
Gly 168 . . . . T T . 0.54 0.51 . . F 0.35 0.91
Thr 169 . . . . T T . 0.54 0.37 . * F 0.65 0.91
Phe 170 . . B . . . . 0.87 0.34 . * F 0.05 0.94
Ser 171 . . . . . T C 0.77 0.34 . * F 0.45 0.94
Pro 172 . . . . . T C 0.17 0.40 . * F 0.45 0.94
Asn 173 . . . . T T . 0.51 0.60 . * F 0.35 0.90
Gly 174 . . . . . T C 0.82 −0.19 . * F 1.20 1.16
Thr 175 . A . . . . C 0.86 −0.57 . * F 1.10 1.30
Leu 176 . A . . . . C 1.16 −0.43 . . F 0.65 0.43
Glu 177 . A B . . . . 1.33 −0.43 . . F 0.45 0.76
Glu 178 . A B . . . . 1.33 −0.36 . . . 0.30 0.72
Cys 179 . A B . . . . 1.37 −0.44 . * . 0.45 1.50
Gln 180 . A . . T . . 1.72 −0.64 * * . 1.15 1.25
His 181 . A . . T . . 1.87 −0.64 . * . 1.15 1.45
Gln 182 . A . . T . . 1.57 −0.07 . * F 1.00 1.45
Thr 183 . . . . T T . 1.28 −0.26 . * F 1.40 1.12
Lys 184 . . . . T T . 1.13 0.26 . * F 0.65 0.87
Cys 185 . . . . T T . 0.28 0.44 . * . 0.20 0.41
Ser 186 . . . . T T . −0.00 0.69 . . . 0.20 0.21
Trp 187 . . B B . . . 0.04 0.69 . * . −0.60 0.15
Leu 188 . . B B . . . −0.23 0.69 * . . −0.60 0.57
Val 189 . . B B . . . −0.62 0.61 * . . −0.60 0.43
Thr 190 . . B B . . . −0.54 0.66 . . . −0.39 0.40
Lys 191 . . B B . . . −0.59 0.24 . . F 0.27 0.50
Ala 192 . . B . . . . −0.61 −0.01 . . F 1.28 0.66
Gly 193 . . . . . T C −0.10 −0.17 . . F 1.89 0.66
Ala 194 . . . . . T C 0.46 −0.27 . . F 2.10 0.44
Gly 195 . . . . . T C 0.47 0.11 . . F 1.29 0.59
Thr 196 . . . . . T C 0.39 −0.00 . . F 1.68 0.80
Ser 197 . . . . . . C 0.69 0.07 . . F 0.82 1.07
Ser 198 . . . . . T C 0.18 0.49 . . F 0.51 1.14
Ser 199 . . . . . T C 0.48 0.70 . . F 0.15 0.59
His 200 . . . . T T . 0.53 1.13 . . . 0.20 0.46
Trp 201 . . B . . T . 0.14 1.66 . . . −0.20 0.36
Val 202 . . B B . . . −0.37 2.06 . . . −0.60 0.23
Trp 203 . . B B . . . −0.37 2.36 . . . −0.60 0.14
Trp 204 . . B B . . . −0.41 2.24 . . . −0.60 0.18
Phe 205 . . B B . . . −0.68 1.76 . . . −0.60 0.24
Leu 206 . . . . . T C −1.20 1.50 . . . 0.00 0.31
Ser 207 . . . . . T C −1.20 1.27 . * F 0.15 0.24
Gly 208 . . . . T T . −1.80 1.00 . . F 0.35 0.21
Ser 209 . . . . . T C −2.37 0.90 . * F 0.15 0.17
Leu 210 . . . B . . C −2.56 0.86 . . . −0.40 0.10
Val 211 . . B B . . . −2.60 1.16 . * . −0.60 0.07
Ile 212 . . B B . . . −2.97 1.37 . . . −0.60 0.04
Val 213 . . B B . . . −2.92 1.56 . . . −0.60 0.02
Ile 214 . . B B . . . −2.93 1.26 . . . −0.60 0.04
Val 215 . . B B . . . −2.98 1.10 . . . −0.60 0.09
Cys 216 . . B B . . . −2.47 1.06 . . . −0.60 0.09
Ser 217 . . B B . . . −2.39 0.84 . . . −0.60 0.13
Thr 218 . . B B . . . −2.42 0.84 . . . −0.60 0.14
Val 219 . . B B . . . −2.42 0.89 . . . −0.60 0.19
Gly 220 . . B B . . . −2.23 1.00 . . . −0.60 0.10
Leu 221 . . B B . . . −2.42 1.19 . * . −0.60 0.04
Ile 222 . . B B . . . −2.08 1.34 * . . −0.60 0.04
Ile 223 . . B B . . . −1.66 0.70 * * . −0.60 0.07
Cys 224 . . B B . . . −0.69 0.27 . . . −0.30 0.18
Val 225 . . B B . . . −0.30 −0.41 . . . 0.30 0.49
Lys 226 . . B B . . . 0.30 −1.10 * * F 1.24 1.40
Arg 227 . . B . . . . 1.30 −1.36 * * F 1.78 4.04
Arg 228 . . . . T . . 1.84 −1.93 . * F 2.52 10.65
Lys 229 . . . . . T C 2.51 −2.14 . * F 2.86 5.27
Pro 230 . . . . T T . 2.51 −2.14 . * F 3.40 4.49
Arg 231 . . . . T T . 1.61 −1.50 . * F 3.06 1.70
Gly 232 . . . . T T . 1.54 −0.86 * * F 2.57 0.63
Asp 233 . . B B . . . 0.58 −0.86 * . F 1.43 0.82
Val 234 . . B B . . . −0.36 −0.64 * * F 1.09 0.31
Val 235 . . B B . . . −1.00 0.04 . * . −0.30 0.22
Lys 236 . . B B . . . −1.41 0.26 * * . −0.30 0.10
Val 237 . . B B . . . −1.92 0.64 * * . −0.60 0.18
Ile 238 . . B B . . . −1.92 0.64 * . . −0.60 0.18
Val 239 . . B B . . . −0.96 0.40 * * . −0.30 0.15
Ser 240 . . B B . . . −0.06 0.40 * * . −0.08 0.40
Val 241 . . B B . . . 0.01 −0.24 * * . 0.89 1.15
Gln 242 . . B B . . . 0.87 −0.93 * . F 1.56 3.03
Arg 243 . . . B . . C 1.76 −1.17 . . F 1.98 3.91
Lys 244 . . . B . . C 2.02 −1.56 . * F 2.20 9.13
Arg 245 . A . . . . C 2.32 −1.70 . * F 1.98 5.33
Gln 246 . A . . . . C 2.83 −2.10 . * F 1.76 4.71
Glu 247 . A . . . . C 2.83 −1.67 . * F 1.54 2.33
Ala 248 . A . . . . C 2.13 −1.67 . * F 1.32 2.06
Glu 249 . A . . . . C 1.78 −1.17 . * F 1.10 1.20
Gly 250 . A . . . . C 0.81 −1.09 * * F 1.10 1.00
Glu 251 A A . B . . . −0.08 −0.44 . * F 0.45 0.74
Ala 252 A A . B . . . −0.08 −0.26 . * F 0.45 0.30
Thr 253 A A . B . . . −0.08 −0.26 * * . 0.30 0.52
Val 254 A A . B . . . −0.89 −0.19 * . . 0.30 0.30
Ile 255 . A B B . . . −0.54 0.50 . . . −0.60 0.25
Glu 256 . A B B . . . −1.13 0.40 . . . −0.30 0.30
Ala 257 . A B . . . . −0.76 0.41 * . . −0.60 0.41
Leu 258 . A B . . . . −0.66 0.20 * . . −0.30 0.89
Gln 259 . A . . . . C 0.20 −0.06 * * . 0.78 0.80
Ala 260 . A . . . . C 0.23 −0.06 * * F 1.36 1.32
Pro 261 . . . . . T C −0.08 0.09 * * F 1.44 1.19
Pro 262 . . . . T T . 0.20 −0.11 * * F 2.37 0.99
Asp 263 . . . . T T . 0.16 −0.03 * * F 2.80 1.42
Val 264 . . B . . T . −0.43 0.11 * * F 1.37 0.68
Thr 265 . . B B . . . −0.70 0.19 * . F 0.69 0.44
Thr 266 . . B B . . . −0.49 0.40 . . . 0.26 0.20
Val 267 . . B B . . . −0.28 0.40 . . . −0.02 0.46
Ala 268 . . B B . . . −0.59 −0.24 . * . 0.30 0.55
Val 269 . . B B . . . −0.62 −0.24 . . . 0.30 0.55
Glu 270 . . B B . . . −0.52 −0.04 . . F 0.45 0.52
Glu 271 . . B B . . . −0.51 −0.26 * . F 0.45 0.80
Thr 272 . . B B . . . −0.36 −0.37 * . F 0.60 1.44
Ile 273 . . B B . . . −0.08 −0.23 * . F 0.45 0.72
Pro 274 . . B B . . . 0.43 0.26 * * F −0.15 0.60
Ser 275 . . . B T . . 0.54 0.69 * * F −0.05 0.41
Phe 276 . . . B T . . 0.24 0.20 * * F 0.40 1.15
Thr 277 . . . B T . . 0.34 −0.10 * . F 0.85 1.00
Gly 278 . . . . T . . 1.23 −0.10 * * F 1.45 1.15
Arg 279 . . . . . . C 1.41 −0.09 * * F 1.50 2.14
Ser 280 . . . . . T C 1.32 −0.37 * * F 1.95 2.01
Pro 281 . . . . . T C 1.63 −0.43 * * . 2.05 2.60
Asn 282 . . . . T T . 1.56 −0.43 * . . 2.50 1.70
His 283 . . . . . T C 1.51 −0.00 * . . 2.05 1.62

TABLE III
Res Position I II III IV V VI VII VIII IX X XI XII XIII XIV
Met 1 . . B . . . . 0.58 −0.24 . . . 1.15 1.53
Glu 2 . . B . . . . 0.97 −0.24 * . . 1.40 1.19
Pro 3 . . . . . T C 1.07 −0.67 * . . 2.35 1.55
Pro 4 . . . . T T . 1.11 −0.19 * . . 2.50 1.65
Gly 5 . . . . T T . 1.29 −0.37 * . F 2.25 0.94
Asp 6 . . . . T T . 1.68 0.06 * . F 1.40 0.94
Trp 7 . . . . T . . 1.47 0.06 * . F 0.95 0.94
Gly 8 . . . . . . C 1.39 0.06 * . F 0.65 1.47
Pro 9 . . . . . T C 1.71 0.54 * . F 0.15 0.93
Pro 10 . . . . . T C 1.76 0.54 * . F 0.30 1.73
Pro 11 . . . . T T . 1.44 0.01 * . F 0.80 2.34
Trp 12 . . . . T T . 1.52 0.07 * * F 0.80 2.18
Arg 13 . . . . T . . 1.98 0.07 * * F 0.94 2.18
Ser 14 . . . . . . C 1.88 −0.36 * * F 1.68 2.77
Thr 15 . . . . . T C 2.09 −0.30 * * F 2.22 3.80
Pro 16 . . . . . T C 1.44 −1.21 * * F 2.86 3.24
Arg 17 . . . . T T . 0.92 −0.57 . * F 3.40 1.79
Thr 18 . . B . . T . 0.92 −0.27 . * F 2.36 1.02
Asp 19 . . B B . . . 0.41 −0.76 * . F 1.92 1.30
Val 20 . . B B . . . −0.13 −0.50 * . . 1.28 0.55
Leu 21 . . B B . . . −0.73 0.14 * . . 0.04 0.28
Arg 22 . . B B . . . −1.09 0.34 * * . −0.30 0.14
Leu 23 . . B B . . . −1.59 1.10 * * . −0.60 0.29
Val 24 . . B B . . . −1.90 1.14 * * . −0.60 0.29
Leu 25 . . B B . . . −1.74 0.94 * * . −0.60 0.22
Tyr 26 . . B B . . . −1.74 1.73 * * . −0.60 0.23
Leu 27 . . B B . . . −2.20 1.73 * * . −0.60 0.25
Thr 28 . . B B . . . −1.98 1.51 . . . −0.60 0.30
Phe 29 . . B B . . . −1.33 1.33 . . . −0.60 0.19
Leu 30 . . B B . . . −1.19 1.00 . . . −0.60 0.37
Gly 31 . . B B . . . −1.19 0.89 . . . −0.60 0.14
Ala 32 . . B . . T . −0.97 1.16 . . . −0.20 0.25
Pro 33 . . . . T T . −0.87 0.87 . . . 0.20 0.30
Cys 34 . . . . T T . −0.76 0.61 . . . 0.20 0.47
Tyr 35 . . B . . T . −0.76 0.69 . . . −0.20 0.47
Ala 36 . . B . . . . −0.62 0.87 . . . −0.40 0.25
Pro 37 . . B . . . . −0.33 0.87 . . . −0.40 0.72
Ala 38 . . B . . . . −0.79 0.69 . . . −0.14 0.62
Leu 39 . . B . . T . −0.08 0.50 . . . 0.32 0.33
Pro 40 . . B . . T . 0.17 0.00 . . F 1.03 0.42
Ser 41 . . . . T T . 0.76 −0.43 . . F 2.29 0.73
Cys 42 . . B . . T . 0.97 −0.93 . . F 2.60 1.47
Lys 43 . A B . . . . 1.31 −1.61 . . F 1.94 1.65
Glu 44 . A B . . . . 1.91 −1.29 . . F 1.68 1.93
Asp 45 . A . . T . . 1.27 −1.24 . . F 1.82 5.56
Glu 46 . A B . . . . 1.22 −1.17 . . F 1.16 2.06
Tyr 47 . A B . . . . 1.59 −0.74 . . . 0.75 1.18
Pro 48 . . . . T . . 1.54 −0.36 . . . 0.90 0.95
Val 49 . . . . T . . 0.88 −0.36 . . F 1.05 0.95
Gly 50 . . . . T T . 0.21 0.21 . . F 0.65 0.32
Ser 51 . . . . T T . 0.00 0.03 . . F 0.65 0.11
Glu 52 . . . . T T . 0.29 0.03 * . F 0.90 0.23
Cys 53 . . B . . T . −0.17 −0.61 . . . 1.50 0.47
Cys 54 . . B . . T . 0.39 −0.47 . . . 1.45 0.19
Pro 55 . . . . T T . 0.52 −0.47 * . . 2.10 0.15
Lys 56 . . . . T T . 0.48 −0.04 * . F 2.50 0.42
Cys 57 . . . . T T . 0.23 −0.19 * * F 2.25 0.78
Ser 58 . . . . . T C 1.01 0.00 * * F 1.20 0.79
Pro 59 . . . . T T . 0.82 −0.43 . * F 1.75 0.77
Gly 60 . . . . T T . 1.08 0.21 . * F 1.05 1.07
Tyr 61 . . B . . T . 1.03 −0.36 . * . 0.85 1.60
Arg 62 . A B . . . . 1.11 −0.74 * * . 0.75 1.79
Val 63 . A B . . . . 0.74 −0.67 * * . 0.75 1.83
Lys 64 . A B . . . . 0.61 −0.53 * . . 0.60 0.63
Glu 65 . A B . . . . 0.96 −0.86 * * . 0.60 0.32
Ala 66 . A B . . . . 0.39 −0.86 * * . 0.60 0.74
Cys 67 . A B . . . . −0.03 −0.81 * * . 0.60 0.30
Gly 68 . A . . T . . 0.48 −0.33 * . . 0.70 0.25
Glu 69 . A . . T . . 0.12 0.10 * * F 0.25 0.25
Leu 70 . . . B T . . −0.73 0.09 * * F 0.25 0.67
Thr 71 . . . B T . . −0.81 0.16 * . F 0.25 0.50
Gly 72 . . . B T . . −0.14 0.30 * . F 0.25 0.16
Thr 73 . . . B T . . −0.01 0.30 * . F 0.25 0.33
Val 74 . . B B . . . −0.68 0.04 * . . −0.30 0.35
Cys 75 . . B B . . . −0.08 0.13 . . . −0.30 0.19
Glu 76 . . B B . . . 0.02 0.13 . . . −0.23 0.20
Pro 77 . . . . T . . 0.02 0.07 . . F 0.59 0.42
Cys 78 . . . . . . C 0.02 −0.14 . . F 1.06 0.78
Pro 79 . . . . . T C 0.63 −0.23 * . F 1.33 0.65
Pro 80 . . . . T T . 0.41 0.53 . . F 0.70 0.66
Gly 81 . . . . T T . −0.18 0.79 * . F 0.63 0.86
Thr 82 . . B . . T . 0.00 0.71 * . F 0.16 0.56
Tyr 83 . . B . . . . −0.14 0.79 . . . −0.26 0.49
Ile 84 . . B . . . . 0.07 1.04 . . . −0.33 0.41
Ala 85 . . B . . . . −0.07 1.01 . . . −0.40 0.46
His 86 . . B . . T . −0.53 0.96 . . . −0.20 0.29
Leu 87 . . B . . T . −0.52 0.89 * . . −0.20 0.34
Asn 88 . . . . T T . −0.23 0.59 * . . 0.20 0.45
Gly 89 . . . . T T . −0.01 0.09 * . F 0.65 0.66
Leu 90 . . . . T . . −0.23 0.16 * . F 0.45 0.43
Ser 91 . . . . T T . −0.20 0.16 * . F 0.65 0.22
Lys 92 . . . . T T . −0.06 0.16 * . . 0.50 0.39
Cys 93 . . B . . T . −0.06 0.30 * . . 0.10 0.25
Leu 94 . . B . . T . −0.31 0.01 * . . 0.10 0.33
Gln 95 . A B . . . . −0.17 0.24 * . . −0.30 0.16
Cys 96 . A B . . . . 0.13 0.81 * . . −0.60 0.16
Gln 97 . A B . . . . −0.12 0.24 * . . −0.30 0.33
Met 98 . A B . . . . 0.54 −0.01 . . . 0.61 0.29
Cys 99 . A B . . . . 0.47 −0.41 . . . 0.92 0.91
Asp 100 . . B . . T . 0.12 −0.30 . . . 1.63 0.37
Pro 101 . . . . T T . 0.49 −0.27 . . F 2.49 0.37
Asp 102 . . . . T T . 0.28 −0.50 . . F 3.10 0.92
Ile 103 . . . . T T . 0.21 −0.64 . . F 2.79 0.85
Gly 104 . . . . T . . 0.88 −0.07 . * F 1.98 0.29
Ser 105 . . B . . T . 0.07 −0.50 . * F 1.77 0.29
Pro 106 . . B . . T . 0.39 0.19 . * F 0.56 0.35
Cys 107 . . B . . T . 0.04 −0.50 * * F 1.46 0.69
Asp 108 . . B . . T . 1.04 −0.50 . * F 1.77 0.51
Leu 109 . . B . . . . 1.04 −0.89 . * F 1.88 0.64
Arg 110 . . B . . . . 1.31 −0.89 . * F 2.34 1.19
Gly 111 . . . . T T . 0.71 −0.96 . * F 3.10 0.97
Arg 112 . . . . T T . 1.38 −0.27 . * F 2.49 0.97
Gly 113 . . . . . T C 0.79 −0.96 . * F 2.28 0.85
His 114 . . . . . T C 1.26 −0.46 . * . 1.52 0.87
Leu 115 . A B . . . . 0.56 −0.46 * * . 0.61 0.44
Glu 116 . A B . . . . 0.87 0.04 * * . −0.30 0.45
Ala 117 . A B . . . . −0.06 0.11 * * . −0.30 0.45
Gly 118 . A B . . . . −0.01 0.30 . * . −0.30 0.45
Ala 119 . A . . T . C −0.19 −0.00 . * . 0.70 0.35
His 120 . A . . . . C 0.28 0.43 . * . −0.06 0.53
Leu 121 . A . . . . C 0.39 0.36 . . . 0.58 0.53
Ser 122 . . . . . T C 0.98 −0.07 . . F 2.22 1.03
Pro 123 . . . . . T C 1.37 −0.17 . . F 2.56 1.32
Gly 124 . . . . T T . 1.61 −0.67 . * F 3.40 3.19
Arg 125 . . . . T T . 1.64 −0.93 . * F 3.06 2.35
Gln 126 . . . . T . . 2.24 −1.31 . * F 2.82 2.64
Lys 127 . . . . T . . 2.54 −1.31 . * F 2.78 4.12
Gly 128 . . . . . . C 2.54 −1.74 . * F 2.54 3.51
Glu 129 . . . . . T C 2.89 −1.31 * * F 2.70 3.14
Pro 130 . . . . . T C 1.92 −1.71 * * F 3.00 2.72
Asp 131 . . . . . T C 1.33 −1.07 . * F 2.70 2.04
Pro 132 . . . . . T C 0.59 −1.00 . * F 2.40 1.19
Glu 133 . A B . . . . 0.93 −0.21 . * . 0.90 0.67
Val 134 . A B . . . . 0.63 −0.64 . * . 0.90 0.69
Ala 135 A A . . . . . 0.03 −0.26 * . . 0.30 0.60
Phe 136 A A . . . . . −0.27 0.00 . . . −0.30 0.28
Glu 137 A A . . . . . −0.64 0.39 . . . −0.30 0.51
Ser 138 A A . . . . . −0.64 0.24 . . . −0.30 0.51
Leu 139 . A . . . . C 0.00 −0.26 . . . 0.65 1.03
Ser 140 . A . . . . C −0.27 −0.61 * . F 0.95 0.92
Ala 141 . A . . . . C 0.40 0.03 * . F 0.05 0.51
Glu 142 . A B . . . . −0.19 0.14 . . F −0.15 0.84
Pro 143 A A . . . . . −0.48 −0.04 . . . 0.30 0.63
Val 144 . A B . . . . 0.33 0.07 . . . −0.30 0.63
His 145 . A B . . . . 0.29 −0.03 . . . 0.30 0.59
Ala 146 . . B . . T . 0.58 0.40 * . . −0.20 0.38
Ala 147 . . . . T T C −0.28 0.36 * . . 0.50 0.68
Asn 148 . . . . T T . −0.28 0.36 . . F 0.65 0.37
Gly 149 . . . . T T . −0.23 0.29 . * F 0.65 0.57
Ser 150 . . . . . . C −0.20 0.47 . * F −0.05 0.46
Val 151 . . . . . . C 0.18 −0.03 . * F 0.85 0.50
Pro 152 . . B . . . . 0.73 0.00 . * . −0.10 0.78
Leu 153 . A B . . . . 0.14 0.07 * * . −0.30 0.79
Glu 154 . A B . . . . 0.60 0.19 * * . −0.15 1.08
Pro 155 . A B . . . . 0.09 −0.46 * * . 0.45 1.36
His 156 . A B . . . . 0.64 −0.20 * * . 0.45 1.36
Ala 157 A A . . . . . 0.26 −0.50 * * . 0.45 1.05
Arg 158 . A B . . . . 0.48 0.11 * * . −0.30 0.68
Leu 159 . A B . . . . 0.18 0.19 . * . −0.30 0.50
Ser 160 . A B . . . . −0.20 0.07 . * . −0.30 0.66
Met 161 . A B . . . . −0.38 0.07 . * . −0.30 0.34
Ala 162 . A B . T . . −0.46 0.50 * * . −0.20 0.64
Ser 163 . . B . . . . −0.91 0.39 * . . −0.10 0.26
Ala 164 . . . . . T C −0.10 0.43 . . . 0.00 0.26
Pro 165 . . . . T T . −0.39 0.21 . . . 0.50 0.44
Cys 166 . . . . T T . −0.13 0.21 . . . 0.50 0.33
Gly 167 . . . . T T . −0.36 0.26 . . F 0.65 0.33
Gln 168 . A B . . . . −0.09 0.44 . . F −0.45 0.17
Ala 169 . A B . . . . −0.31 0.51 . * . −0.60 0.44
Gly 170 . A B . . . . 0.01 0.63 * * . −0.60 0.37
Leu 171 . A B . . . . 0.68 0.20 * * . −0.30 0.42
His 172 . A B . . . . 1.13 −0.20 * * . 0.30 0.69
Leu 173 . A B . . . . 0.54 −0.70 * * . 1.09 1.36
Arg 174 . A B . . . . 1.13 −0.63 . * . 1.43 1.66
Asp 175 . A B . . . . 1.13 −1.31 . * F 1.92 2.04
Arg 176 . . B . . T . 1.63 −1.39 . * F 2.66 2.45
Ala 177 . . . . T T . 1.46 −1.59 . * F 3.40 1.81
Asp 178 . . . . T T . 1.92 −1.16 . * F 3.06 1.67
Gly 179 . . . . . T C 1.47 −0.73 * * F 2.55 0.85
Thr 180 . . . . . T C 1.58 −0.30 * * F 2.09 0.83
Pro 181 . . . . . T C 0.88 −0.80 * * F 2.23 0.97
Gly 182 . . . . T T . 1.08 −0.30 * * F 1.97 0.99
Gly 183 . . . . . T C 0.69 −0.30 * * . 1.80 0.88
Arg 184 . . B . . . . 0.64 −0.36 * . . 1.22 0.73
Ala 185 . . B . . . . 0.57 −0.36 * . . 1.04 0.94

TABLE IV
Res Position I II III IV V VI VII VIII IX X XI XII XIII XIV
Met 1 . . B . . . . −0.42 0.50 . . . −0.40 0.44
Leu 2 . . B . . . . −0.38 0.46 . . . −0.40 0.46
Gly 3 . . B . . T . −0.02 0.46 . . . −0.20 0.35
Thr 4 . . . . . T C −0.44 0.53 . . . 0.00 0.49
Ser 5 . . . . . T C −0.91 0.60 . . F 0.15 0.49
Gly 6 . . B . . T . −0.60 0.56 . . F −0.05 0.37
His 7 . . B B . . . −0.60 1.04 . . . −0.60 0.27
Leu 8 . . B B . . . −0.56 1.24 . . . −0.60 0.16
Val 9 . . B B . . . −0.24 1.24 . . . −0.60 0.22
Trp 10 . . B B . . . −0.29 1.21 . . . −0.60 0.28
Leu 11 . . B B . . . −0.64 1.14 . . . −0.60 0.34
Ser 12 . . B . . T . −0.91 1.24 . . F −0.05 0.40
Gln 13 . . B . . T . −0.91 0.99 . . F −0.05 0.50
Gly 14 . . . . T T . −0.64 0.76 . . F 0.35 0.50
Phe 15 . . B . . T . −0.70 0.57 * * . −0.20 0.38
Ser 16 . . B . . . . 0.22 0.61 * * . −0.40 0.22
Leu 17 . . B . . . . 0.31 0.21 * * . −0.10 0.43
Ala 18 . . B . . . . −0.03 0.21 * * . 0.24 0.77
Gly 19 . . . . . . C 0.01 −0.14 * * F 1.53 0.57
Arg 20 . . . . . T C 0.41 −0.14 * * F 2.07 0.92
Pro 21 . . . . T T . 0.50 −0.44 * * F 2.76 1.22
Gly 22 . . . . T T . 1.02 −0.51 * * F 3.40 1.91
Ser 23 . . . . . T C 1.40 −0.03 * . F 2.56 1.02
Ser 24 . . . . . T C 0.89 0.40 . * F 1.32 1.02
Pro 25 . . . . . T C 0.78 0.61 . * F 0.83 0.77
Trp 26 . . B . . T . 0.40 0.19 . * F 0.59 0.96
Pro 27 . . B . . T . −0.11 0.30 . * . 0.10 0.72
Val 28 . . B B . . . −0.62 0.56 . * . −0.60 0.35
Asp 29 . . B B . . . −0.91 0.81 . * . −0.60 0.27
Ala 30 . . B B . . . −1.37 0.40 . * . −0.60 0.18
Val 31 . . B B . . . −1.42 0.54 . * . −0.60 0.13
Leu 32 . . B B . . . −1.50 0.33 . . . −0.30 0.08
Ala 33 . . B . . T . −1.31 1.24 . . . −0.20 0.08
Cys 34 . . . . T T . −1.52 1.31 . . . 0.20 0.06
Gly 35 . . . . T T . −1.28 1.10 . . . 0.20 0.11
Trp 36 . . . . T T . −1.23 0.84 . . . 0.20 0.10
Cys 37 . . B . . T . −0.46 1.03 . * . −0.20 0.16
Pro 38 . . . . T T . −0.72 0.96 . . . 0.20 0.22
Gly 39 . . . . T T . −0.27 1.17 . * . 0.20 0.16
Leu 40 . . B . . T . −0.13 0.69 . . . −0.20 0.45
His 41 . . B . . . . −0.66 0.54 . . . −0.40 0.45
Val 42 . . B . . . . −0.29 0.80 . . . −0.40 0.38
Pro 43 . . B . . . . −0.29 0.76 . . F −0.25 0.61
Pro 44 . . . . T . . −0.24 0.50 . . F 0.15 0.70
Leu 45 . . . . T . . 0.27 0.39 . . F 0.60 1.26
Ser 46 . . . . . T C 0.01 0.13 . . F 0.60 1.09
Pro 47 . . . . . T C 0.56 0.61 . . F 0.15 0.74
Ser 48 . . . . T T . 0.56 0.67 . . F 0.50 1.29
Ser 49 . . . . T T . 0.18 0.41 . . F 0.50 1.49
Trp 50 . . B . . . . 0.39 0.53 . . F −0.25 0.98
Thr 51 . . B . . . . 0.34 0.71 . . F −0.25 0.72
Pro 52 . . B . . . . −0.26 0.76 . * . −0.40 0.53
Ala 53 . A B . . . . 0.16 1.06 . * . −0.60 0.42
Met 54 . A B . . . . −0.13 0.14 . * . −0.30 0.57
Gly 55 . A B . . . . −0.14 0.16 * * . −0.30 0.37
Leu 56 . A B . . . . 0.28 0.11 * * . −0.30 0.49
Arg 57 . A B . . . . 0.49 −0.39 * * . 0.64 0.97
Ala 58 . A B . . . . 0.41 −0.60 * * . 1.43 1.58
Ser 59 . . . . T T . 0.71 −0.46 * * F 2.42 1.03
Arg 60 . . . . T T . 1.17 −0.76 * * F 2.91 0.70
Asn 61 . . . . T T . 1.67 −0.76 * * F 3.40 1.36
Cys 62 . . . . T T . 1.56 −0.77 * * F 3.06 1.47
Ser 63 . . . . T . . 2.14 −1.16 * * F 2.52 1.30
Arg 64 . A . . T . . 1.86 −0.76 * * F 1.98 1.30
Thr 65 . A . . T . . 0.89 −0.66 * * F 1.64 2.44
Glu 66 . A . . T . . 0.22 −0.59 * . F 1.30 1.35
Asn 67 . A B . . . . 0.54 −0.40 * * F 0.45 0.37
Ala 68 . A B . . . . 0.18 0.03 . * . −0.30 0.25
Val 69 . A . . T . . −0.23 0.11 . * . 0.10 0.08
Cys 70 . . . . T . . −0.13 0.50 . . . 0.00 0.07
Gly 71 . . . . T . . −0.48 0.53 . . . 0.00 0.10
Cys 72 . . . . T . . −0.51 0.46 . . . 0.00 0.13
Ser 73 . . . . . T C −0.62 0.31 . . . 0.30 0.34
Pro 74 . . . . T T . −0.43 0.53 . * F 0.35 0.30
Gly 75 . . . . T T . −0.66 0.67 . . . 0.20 0.30
His 76 . . B . . T . −1.17 0.79 . . . −0.20 0.16
Phe 77 . . B B . . . −0.50 1.04 . . . −0.60 0.07
Cys 78 . . B B . . . −0.20 1.01 . . . −0.32 0.13
Ile 79 . . B B . . . −0.33 0.59 . * . −0.04 0.16
Val 80 . . B . . T . 0.01 0.51 . * . 0.64 0.18
Gln 81 . . . . T T . 0.01 −0.27 . . F 2.37 0.57
Asp 82 . . . . T T . 0.04 −0.34 * . F 2.80 1.11
Gly 83 . . . . T T . 0.12 −0.46 * . F 2.37 0.80
Asp 84 . A . . T . . 0.42 −0.60 * . F 1.99 0.47
His 85 . A . . T . . 0.61 −0.50 . * . 1.56 0.28
Cys 86 . A B . . . . 0.72 0.07 . * . −0.02 0.15
Ala 87 . A B . . . . 0.13 −0.36 . * . 0.30 0.18
Ala 88 . A B . . . . 0.23 0.14 . * . −0.30 0.13
Cys 89 . A B . . . . −0.36 0.40 . * . −0.30 0.39
Arg 90 . A B . . . . −0.63 0.33 . * . −0.30 0.39
Ala 91 . A B . . . . −0.27 0.31 . * . −0.30 0.56
Tyr 92 . . B . . . . 0.02 0.20 . * . 0.05 1.39
Ala 93 . . B . . . . 0.40 0.01 . * . −0.10 0.95
Thr 94 . . . . T . . 0.72 0.44 . * F 0.30 1.45
Ser 95 . . . . . . C 0.61 0.37 * * F 0.25 0.92
Ser 96 . . . . . T C 1.31 0.01 * * F 0.60 1.57
Pro 97 . . . . . T C 0.70 −0.49 * * F 1.20 2.14
Gly 98 . . . . T T . 1.29 −0.33 * * F 1.40 1.18
Gln 99 . . B . . T . 1.64 −0.31 * . F 1.00 1.53
Arg 100 . . B . . . . 1.60 −0.70 * . F 1.40 1.98
Val 101 . . B . . . . 1.56 −0.70 * . F 1.70 1.98
Gln 102 . . B . . T . 1.46 −0.70 * . F 2.20 1.13
Lys 103 . . B . . T . 1.80 −0.61 * . F 2.35 0.83
Gly 104 . . . . . T C 1.50 −0.61 * * F 3.00 1.94
Gly 105 . . . . . T C 1.39 −0.87 * * F 2.70 1.50
Thr 106 . . . . . . C 2.24 −0.87 * . F 2.45 1.30
Glu 107 . . . . . . C 1.93 −0.87 * . F 2.40 2.20
Ser 108 . . B . T T . 1.08 −0.81 * * F 2.75 3.20
Gln 109 . . . . T T . 0.76 −0.56 * . F 2.70 1.83
Asp 110 . . . . T T . 1.10 −0.47 . . F 2.50 0.57
Thr 111 . . B . . T . 1.41 −0.07 . . F 1.85 0.73
Leu 112 . . . . T . . 0.74 −0.06 . . . 1.65 0.68
Cys 113 . . . . T . . 0.83 0.11 * . . 1.05 0.22
Gln 114 . . B . . . . 0.94 0.54 * * . 0.35 0.23
Asn 115 . . B . . . . 0.60 0.06 * * . 0.65 0.56
Cys 116 . . B . . T . 0.70 −0.20 * * F 2.00 1.03
Pro 117 . . . . T T . 1.21 −0.34 * . F 2.50 0.92
Arg 118 . . . . T T . 1.07 −0.36 * . F 2.25 0.76
Gly 119 . . . . . T C 0.86 −0.07 * . F 1.95 1.17
Pro 120 . . . . T . . 0.26 −0.21 . . F 1.70 1.17
Ser 121 . . . . . . C 0.58 −0.03 . . F 1.10 0.59
Leu 122 . . B . . . . 0.58 0.40 . . . −0.10 0.59
Pro 123 . . B . . . . 0.18 0.40 * * . −0.10 0.59
Met 124 . . B . . . . 0.63 0.89 * . . −0.40 0.47
Gly 125 . . B . . T . 0.84 0.50 * . . −0.05 1.11
Pro 126 . . . . T T . 0.29 0.21 * . F 0.80 1.15
Trp 127 . . . . T T . 0.80 0.43 * . F 0.35 0.86
Arg 128 . . B . . T . 0.70 0.20 * * F 0.40 1.17
Asn 129 . . B . . . . 1.41 0.26 * * F 0.20 1.09
Val 130 . . B . . . . 1.54 −0.17 * * F 1.11 2.03
Ser 131 . . B . . . . 1.46 −0.66 * . F 1.72 1.60
Thr 132 . . . . . . C 1.79 −0.27 * . F 1.93 1.34
Arg 133 . . . . . T C 1.29 −0.67 * . F 2.74 3.60
Pro 134 . . . . T T . 0.90 −0.89 * * . 3.10 3.43
Ser 135 . . . . T T . 1.37 −0.84 * * . 2.79 3.04
Lys 136 . . . . T T . 1.28 −0.90 * . . 2.48 1.98

Among highly preferred fragments in this regard are those that comprise, or alternatively consist of, regions of TR2 receptors that combine several structural features, such as several of the features set out above. Polynucleotides encoding these polypeptides are also encompassed by the invention.

As one of skill in the art will appreciate, TR2 polypeptides of the present invention and epitope-bearing fragments thereof can be combined with heterologous polypeptide sequences. For example, the polypeptides of the present invention may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM) or portions thereof (CH1, CH2, CH3, and any combination thereof, including both entire domains and portions thereof), resulting in chimeric polypeptides. These fusion proteins facilitate purification and show an increased half-life.

The present invention is further directed to isolated polypeptides comprising, or alternatively consisting of, fragments of TR2, TR2-SV1, and TR2-SV2. In particular, the invention provides isolated polypeptides comprising, or alternatively consisting of, the amino acid sequences of a member selected from the group consisting of amino acids −36 to 24, −26 to 34, −16 to 44, −6 to 54, 1 to 60, 11 to 70, 21 to 80, 31 to 90, 41 to 100, 51 to 110, 61 to 120, 71 to 130, 81 to 140, 91 to 150, 101 to 160, 111 to 170, 121 to 180, 131 to 190, 141 to 200, 151 to 210, 161 to 220, 171 to 230, 181 to 240, and 191 to 247 of SEQ ID NO:2, as well as isolated polynucleotides which encode these polypeptides. The invention further provides isolated polypeptides comprising, or alternatively consisting of, the amino acid sequences of a member selected from the group consisting of amino acids −36 to 24, −26 to 34, −16 to 44, −6 to 54, 1 to 60, 11 to 70, 21 to 80, 31 to 90, 41 to 100, 51 to 10, 61 to 120, 71 to 130, 81 to 140, and 91 to 149 of SEQ ID NO:5, as well as isolated polynucleotides which encode these polypeptides. The invention also provides isolated polypeptides comprising, or alternatively consisting of, the amino acid sequences of a member selected from the group consisting of amino acids 1 to 60, 11 to 70, 21 to 80, 31 to 90, 41 to 100, 51 to 110, 61 to 120, 71 to 130, and 81 to 136 of SEQ ID NO:8, as well as isolated polynucleotides which encode these polypeptides.

The present invention is also directed to isolated polypeptides comprising, or alternatively consisting of, domains of TR2, TR2-SV1, and TR2-SV2. In particular, the invention provides polypeptides comprising, or alternatively consisting of, beta-sheet regions of TR2, TR2-SV1, and TR2-SV2 set out in Tables II, III and IV. These polypeptides include polypeptides comprising, or alternatively consisting of, amino acid sequences of a member selected from the group consisting of amino acid residues from about −19 to about −5, amino acid residues from about −18 to about −6, amino acid residues from about −2 to about 4, amino acid residues from about 25 to about 31, amino acid residues from about 46 to about 51, amino acid residues from about 57 to about 71, amino acid residues from about 99 to about 104, amino acid residues from about 151 to about 156, amino acid residues from about 175 to about 191, amino acid residues from about 174 to about 190, amino acid residues from about 197 to about 206, amino acid residues from about 197 to about 208, amino acid residues from about 215 to about 220, amino acid residues from about 228 to about 238, and amino acid residues from about 229 to about 241 of SEQ ID NO:2; amino acid residues from about −19 to about −5, amino acid residues from about −18 to about −6, amino acid residues from about −2 to about 3, amino acid residues from about 26 to about 31, amino acid residues from about 34 to about 40, amino acid residues from about 46 to about 50, amino acid residues from about 57 to about 64, amino acid residues from about 69 to about 74, amino acid residues from about 122 to about 128, and amino acid residues from about 132 to about 140 of SEQ ID NO:5; and amino acid residues from about 6 to about 13, amino acid residues from about 26 to about 33, amino acid residues from about 50 to about 58, and amino acid residues from about 86 to about 93 of SEQ ID NO:8. The invention is further directed to isolated polynucleotides comprising, or alternatively consisting of, nucleic acid molecules which encode the beta-sheet regions set out in Tables II, III and IV, and isolated polypeptides comprising, or alternatively consisting of, amino acid sequences at least 80% identical, and more preferably at least 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to nucleic acid molecules encoding beta-sheet regions of the TR2, TR2-SV1, and TR2-SV2 proteins.

The TR2 receptor proteins of the invention may be in monomers or multimers (i.e., dimers, trimers, tetramers, and higher multimers). Accordingly, the present invention relates to monomers and multimers of the TR2 receptor proteins of the invention, their preparation, and compositions (preferably, pharmaceutical compositions) containing them. In specific embodiments, the polypeptides of the invention are monomers, dimers, trimers or tetramers. In additional embodiments, the multimers of the invention are at least dimers, at least trimers, or at least tetramers.

Multimers encompassed by the invention may be homomers or heteromers. As used herein, the term homomer, refers to a multimer containing only TR2 receptor proteins of the invention (including TR2 receptor fragments, variants, and fusion proteins, as described herein). These homomers may contain TR2 receptor proteins having identical or different polypeptide sequences. In a specific embodiment, a homomer of the invention is a multimer containing only TR2 receptor proteins having an identical polypeptide sequence. In another specific embodiment, a homomer of the invention is a multimer containing TR2 receptor proteins having different polypeptide sequences. In specific embodiments, the multimer of the invention is a homodimer (e.g., containing TR2 receptor proteins having identical or different polypeptide sequences) or a homotrimer (e.g., containing TR2 receptor proteins having identical or different polypeptide sequences). In additional embodiments, the homomeric multimer of the invention is at least a homodimer, at least a homotrimer, or at least a homotetramer.

As used herein, the term heteromer refers to a multimer containing heterologous proteins (i.e., proteins containing only polypeptide sequences that do not correspond to a polypeptide sequences encoded by the TR2 receptor gene) in addition to the TR2 receptor proteins of the invention. In a specific embodiment, the multimer of the invention is a heterodimer, a heterotrimer, or a heterotetramer. In additional embodiments, the heteromeric multimer of the invention is at least a heterodimer, at least a heterotrimer, or at least a heterotetramer.

Multimers of the invention may be the result of hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked, by for example, liposome formation. Thus, in one embodiment, multimers of the invention, such as, for example, homodimers or homotrimers, are formed when proteins of the invention contact one another in solution. In another embodiment, heteromultimers of the invention, such as, for example, heterotrimers or heterotetramers, are formed when proteins of the invention contact antibodies to the polypeptides of the invention (including antibodies to the heterologous polypeptide sequence in a fusion protein of the invention) in solution. In other embodiments, multimers of the invention are formed by covalent associations with and/or between the TR2 receptor proteins of the invention. Such covalent associations may involve one or more amino acid residues contained in the polypeptide sequence of the TR2 receptor proteins (e.g., the polypeptide sequence recited in SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, or SEQ ID NO:26, or the polypeptides encoded by the cDNAs contained in ATCC Deposit Numbers 97059, 97058, or 97057). In one instance, the covalent associations are cross-linking between cysteine residues located within the polypeptide sequences of the proteins which interact in the native (i.e., naturally occurring) polypeptide. In another instance, the covalent associations are the consequence of chemical or recombinant manipulation. Alternatively, such covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in a TR2 receptor fusion protein. In one example, covalent associations are between the heterologous sequence contained in a fusion protein of the invention (see, e.g., U.S. Pat. No. 5,478,925). In a specific example, the covalent associations are between the heterologous sequence contained in a TR2 receptor-Fc fusion protein of the invention (as described herein). In another specific example, covalent associations of fusion proteins of the invention are between heterologous polypeptide sequences from another TNF family ligand/receptor member that is capable of forming covalently associated multimers, such as for example, oseteoprotegerin (see, e.g., International Publication No. WO 98/49305, the contents of which are herein incorporated by reference in its entirety). In another embodiment, two or more TR2 polypeptides of the invention are joined through synthetic linkers (e.g., peptide, carbohydrate or soluble polymer linkers). Examples include, but are not limited to, those peptide linkers described in U.S. Pat. No. 5,073,627 (hereby incorporated by reference). Proteins comprising multiple TR2 polypeptides separated by peptide linkers may be produced using conventional recombinant DNA technology.

Another method for preparing multimer TR2 polypeptides of the invention involves use of TR2 polypeptides fused to a leucine zipper polypeptide sequence. Leucine zipper domains are polypeptides that promote multimerization of the proteins in which they are found. Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., Science 240:1759, (1988)), and have since been found in a variety of different proteins. Among the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize. Examples of leucine zipper domains suitable for producing soluble multimeric TR2 proteins are those described in PCT application WO 94/10308, hereby incorporated by reference. Recombinant fusion proteins comprising a soluble TR2 polypeptide fused to a peptide that dimerizes or trimerizes in solution are expressed in suitable host cells, and the resulting soluble multimeric TR2 is recovered from the culture supernatant using techniques known in the art.

Certain members of the TNF family of proteins are believed to exist in trimeric form (Beutler and Huffel, Science 264:667, 1994; Banner et al., Cell 73:431, 1993). Thus, trimeric TR2 may offer the advantage of enhanced biological activity. Preferred leucine zipper moieties are those that preferentially form trimers. One example is a leucine zipper derived from lung surfactant protein D (SPD), as described in Hoppe et al. (FEBS Letters 344:191, (1994)) and in U.S. patent application Ser. No. 08/446,922, hereby incorporated by reference. Other peptides derived from naturally occurring trimeric proteins may be employed in preparing trimeric TR2.

In another example, proteins of the invention are associated by interactions between Flag® polypeptide sequence contained in Flag®-TR2 or Flag®-TR2 fusion proteins of the invention. In a further embodiment, associations proteins of the invention are associated by interactions between heterologous polypeptide sequence contained in Flag®-TR2 or Flag®-TR2 fusion proteins of the invention and anti-Flag® antibody.

The multimers of the invention may be generated using chemical techniques known in the art. For example, proteins desired to be contained in the multimers of the invention may be chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Additionally, multimers of the invention may be generated using techniques known in the art to form one or more inter-molecule cross-links between the cysteine residues located within the polypeptide sequence of the proteins desired to be contained in the multimer (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Further, proteins of the invention may be routinely modified by the addition of cysteine or biotin to the C-terminus or N-terminus of the polypeptide sequence of the protein and techniques known in the art may be applied to generate multimers containing one or more of these modified proteins (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). Additionally, techniques known in the art may be applied to generate liposomes containing the protein components desired to be contained in the multimer of the invention (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety).

Alternatively, multimers of the invention may be generated using genetic engineering techniques known in the art. In one embodiment, proteins contained in multimers of the invention are produced recombinantly using fusion protein technology described herein or otherwise known in the art (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). In a specific embodiment, polynucleotides coding for a homodimer of the invention are generated by ligating a polynucleotide sequence encoding a polypeptide of the invention to a sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding the translated product of the polypeptide in the reverse orientation from the original C-terminus to the N-terminus (lacking the leader sequence) (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety). In another embodiment, recombinant techniques described herein or otherwise known in the art are applied to generate recombinant polypeptides of the invention which contain a transmembrane domain and which can be incorporated by membrane reconstitution techniques into liposomes (see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated by reference in its entirety).

The present invention encompasses polypeptides comprising, or alternatively consisting of, an epitope of the polypeptide having an amino acid sequence of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, or SEQ ID NO:26, or an epitope of the polypeptide sequence encoded by a polynucleotide sequence contained in the deposited cDNA identified as ATCC Accession No. 97059, 97058 or 97057 or encoded by a polynucleotide that hybridizes to the complement of the polynucleotide sequence of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, or SEQ ID NO:25, or contained in the deposited cDNA identified as ATCC Accession No. 97059, 97058 or 97057 under stringent hybridization conditions or lower stringency hybridization conditions as defined herein. The present invention further encompasses polynucleotide sequences encoding an epitope of a polypeptide sequence of the invention (such as, for example, the sequence disclosed in SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, or SEQ ID NO:26), polynucleotide sequences of the complementary strand of a polynucleotide sequence encoding an epitope of the invention, and polynucleotide sequences which hybridize to the complementary strand under stringent hybridization conditions or lower stringency hybridization conditions defined herein.

The term “epitopes,” as used herein, refers to portions of a polypeptide having antigenic or immunogenic. activity in an animal, preferably a mammal, and most preferably in a human. In a preferred embodiment, the present invention encompasses a polypeptide comprising an epitope, as well as the polynucleotide encoding this polypeptide. An “immunogenic epitope,” as used herein, is defined as a portion of a protein that elicits an antibody response in an animal, as determined by any method known in the art, for example, by the methods for generating antibodies described infra. (See, for example, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983)). The term “antigenic epitope,” as used herein, is defined as a portion of a protein to which an antibody can immunospecifically bind its antigen as determined by any method well known in the art, for example, by the immunoassays described herein. Immunospecific binding excludes non-specific binding but does not necessarily exclude cross-reactivity with other antigens. Antigenic epitopes need not necessarily be immunogenic.

As to the selection of peptides or polypeptides bearing an antigenic epitope (i.e., that contain a region of a protein molecule to which an antibody can bind), it is well known in that art that relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein. See, for instance, Sutcliffe, J. G., Shinnick, T. M., Green, N. and Learner, R. A. (1983) Antibodies that react with predetermined sites on proteins. Science 219:660-666. Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence of a protein, can be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact proteins (i.e., immunogenic epitopes) nor to the amino or carboxyl terminals.

Antigenic epitope-bearing peptides and polypeptides of the invention are therefore useful to raise antibodies, including monoclonal antibodies, that bind specifically to a polypeptide of the invention. See, for instance, Wilson et al., Cell 37:767-778 (1984) at 777.

Antigenic epitopes of the invention preferably contain a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, and, most preferably, between about 15 to about 30 amino acids. In this context “about” includes the particularly recited value and values larger or smaller by several (5, 4, 3, 2, or 1) amino acids. Preferred polypeptides comprising immunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues in length. Antigenic epitopes are useful, for example, to raise antibodies, including monoclonal antibodies, that specifically bind the epitope. Antigenic epitopes can be used as the target molecules in immunoassays. (See, for instance, Wilson et al, Cell 37:767-778 (1984); Sutcliffe et al, Science 219:660-666 (1983)).

Non-limiting examples of antigenic polypeptides or peptides that can be used to generate TR2 receptor-specific antibodies include: a polypeptide comprising, or alternatively consisting of, amino acid residues from about 39 to about 70 in FIG. 1 (amino acid residues 3 to 34 in SEQ ID NO:2); a polypeptide comprising, or alternatively consisting of, amino acid residues from about 106 to about 120 in FIG. 1A-1B (amino acid residues 70 to 84 in SEQ ID NO:2); a polypeptide comprising, or alternatively consisting of, amino acid residues from about 142 to about 189 in FIG. 1A-1B (amino acid residues 106 to 153 in SEQ ID NO:2); a polypeptide comprising, or alternatively consisting of, amino acid residues from about 276 to about 283 in FIG. 1 (amino acid residues 240 to 247 in SEQ ID NO:2); a polypeptide comprising, or alternatively consisting of, amino acid residues from about 39 to about 70 in FIG. 4A-4B (amino acid residues 3 to 34 in SEQ ID NO:5); a polypeptide comprising, or alternatively consisting of, amino acid residues from about 99 to about 136 in FIG. 4A-4B (amino acid residues 63 to 100 in SEQ ID NO:5); a polypeptide comprising, or alternatively consisting of, amino acid residues from about 171 to about 185 in FIG. 4A-4B (amino acid residues 135 to 149 in SEQ ID NO:5); a polypeptide comprising, or alternatively consisting of, amino acid residues from about 56 to about 68 in FIG. 7A-7B (SEQ ID NO:8); and a polypeptide comprising, or alternatively consisting of, amino acid residues from about 93 to about 136 in FIG. 7A-7B (SEQ ID NO:8). In this context “about” includes the particularly recited value and values larger or smaller by several (5, 4, 3, 2, or 1) amino acids. As indicated above, the inventors have determined that the above polypeptide fragments are antigenic regions of the TR2 receptor proteins.

The epitope-bearing peptides and polypeptides of the invention may be produced by any conventional means. Houghten, R. A. (1985) General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids. Proc. Natl. Acad. Sci. USA 82:5131-5135. This “Simultaneous Multiple Peptide Synthesis (SMPS)” process is further described in U.S. Pat. No. 4,631,211 to Houghten et al. (1986).

Similarly, immunogenic epitopes can be used, for example, to induce antibodies according to methods well known in the art. (See, for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol. 66:2347-2354 (1985). A preferred immunogenic epitope includes the secreted protein. The polypeptides comprising one or more immunogenic epitopes may be presented for eliciting an antibody response together with a carrier protein, such as an albumin, to an animal system (such as, for example, rabbit or mouse), or, if the polypeptide is of sufficient length (at least about 25 amino acids), the polypeptide may be presented without a carrier. However, immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting).

Epitope-bearing polypeptides of the present invention may be used to induce antibodies according to methods well known in the art including, but not limited to, in vivo immunization, in vitro immunization, and phage display methods. See, e.g., Sutcliffe et al., supra; Wilson et al., supra, and Bittle et al., J. Gen. Virol., 66:2347-2354 (1985). If in vivo immunization issued, animals may be immunized with free peptide; however, anti-peptide antibody titer may be boosted by coupling the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid. For instance, peptides containing cysteine residues may be coupled to a carrier using a linker such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such as glutaraldehyde. Animals such as, for example, rabbits, rats, and mice are immunized with either free or carrier-coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 micrograms of peptide or carrier protein and Freund's adjuvant or any other adjuvant known for stimulating an immune response. Several booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody that can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface. The titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.

As one of skill in the art will appreciate, and as discussed above, the polypeptides of the present invention comprising an immunogenic or antigenic epitope can be fused to other polypeptide sequences. For example, the polypeptides of the present invention may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CH1, CH2, CH3, or any combination thereof and portions thereof) resulting in chimeric polypeptides. Such fusion proteins may facilitate purification and may increase half-life in vivo. This has been shown for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. See, e.g., EP 394,827; Traunecker et al., Nature, 331:84-86 (1988). IgG Fusion proteins that have a disulfide-linked dimeric structure due to the IgG portion disulfide bonds have also been found to be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone. See, e.g., Fountoulakis et al., J. Biochem., 270:3958-3964 (1995). Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag (e.g., the hemagglutinin (“HA”) tag or flag tag) to aid in detection and purification of the expressed polypeptide. For example, a system described by Janknecht et al. allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-897). In this system, the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of six histidine residues. The tag serves as a matrix-binding domain for the fusion protein. Extracts from cells infected with the recombinant vaccinia virus are loaded onto Ni2+ nitriloacetic acid-agarose column and histidine-tagged proteins can be selectively eluted with imidazole-containing buffers.

Additional fusion proteins of the invention may be generated through the techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as “DNA shuffling”). DNA shuffling may be employed to modulate the activities of polypeptides of the invention, such methods can be used to generate polypeptides with altered activity, as well as agonists and antagonists of the polypeptides. See, generally, U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol. 16(2):76-82 (1998); Hansson et al, J. Mol. Biol. 287:265-76 (1999); and Lorenzo and Blasco, BioTechniques 24(2):308-13 (1998) (each of these patents and publications are hereby incorporated by reference in its entirety). In one embodiment, alteration of polynucleotides corresponding to SEQ ID NO:1 and the polypeptides encoded by these polynucleotides may be achieved by DNA shuffling. DNA shuffling involves the assembly of two or more DNA segments by homologous or site-specific recombination to generate variation in the polynucleotide sequence. In another embodiment, polynucleotides of the invention, or the encoded polypeptides, may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. In another embodiment, one or more components, motifs, sections, parts, domains, fragments, etc., of a polynucleotide coding a polypeptide of the invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.

Proteins of the invention can be chemically synthesized using techniques known in the art (e.g., see Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N.Y., and Hunkapiller, M., et al., Nature 310:105-111 (1984)). For example, a peptide corresponding to a fragment of the TR2 receptor polypeptides of the invention can be synthesized by use of a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the TR2 receptor polypeptide sequence. Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, α-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, γ-Abu, ε-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acids such as β-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).

Non-naturally occurring variants may be produced using art-known mutagenesis techniques, which include, but are not limited to oligonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis, site directed mutagenesis (see, e.g., Carter et al., Nucl. Acids Res. 13:4331 (1986); and Zoller et al., Nucl. Acids Res. 10: 6487 (1982)), cassette mutagenesis (see, e.g., Wells et al., Gene 34:315 (1985)), restriction selection mutagenesis (see, e.g., Wells et al., Philos. Trans. R. Soc. London SerA 317:415 (1986)).

The invention additionally, encompasses TR2 receptor polypeptides which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited to, specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4, acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin; etc.

Additional post-translational modifications encompassed by the invention include, for example, e.g., N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of procaryotic host cell expression. The polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein.

Also provided by the invention are chemically modified derivatives of TR2, TR2-SV1 and TR2-SV2 receptor polypeptides which may provide additional advantages such as increased solubility, stability and circulating time of the polypeptides, or decreased immunogenicity (see U.S. Pat. No. 4,179,337). The chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like. The polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.

The polymer may be of any molecular weight, and may be branched or unbranched. For polyethylene glycol, the preferred molecular weight is between about 1 kDa and about 100 kDa (the term “about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing. Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog). For example, the polyethylene glycol may have an average molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.

As noted above, the polyethylene glycol may have a branched structure. Branched polyethylene glycols are described, for example, in U.S. Pat. No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72 (1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750 (1999); and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999), the disclosures of each of which are incorporated herein by reference.

The polyethylene glycol molecules (or other chemical moieties) should be attached to the protein with consideration of effects on functional or antigenic domains of the protein. There are a number of attachment methods available to those skilled in the art, e.g., EP 0 401 384, herein incorporated by reference (coupling PEG to G-CSF), see also Malik et al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation of GM-CSF using tresyl chloride). For example, polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as, a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule may be bound. The amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues, glutamic acid residues and the C-terminal amino acid residue. Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules. Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.

As suggested above, polyethylene glycol may be attached to proteins via linkage to any of a number of amino acid residues. For example, polyethylene glycol can be linked to a proteins via covalent bonds to lysine, histidine, aspartic acid, glutamic acid, or cysteine residues. One or more reaction chemistries may be employed to attach polyethylene glycol to specific amino acid residues (e.g., lysine, histidine, aspartic acid, glutamic acid, or cysteine) of the protein or to more than one type of amino acid residue (e.g., lysine, histidine, aspartic acid, glutamic acid, cysteine and combinations thereof) of the protein.

One may specifically desire proteins chemically modified at the N-terminus. Using polyethylene glycol as an illustration of the present composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (or peptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein. The method of obtaining the N-terminally pegylated preparation (i.e., separating this moiety from other monopegylated moieties if necessary) may be by purification of the N-terminally pegylated material from a population of pegylated protein molecules. Selective proteins chemically modified at the N-terminus modification may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.

As indicated above, pegylation of the proteins of the invention may be accomplished by any number of means. For example, polyethylene glycol may be attached to the protein either directly or by an intervening linker. Linkerless systems for attaching polyethylene glycol to proteins are described in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992); Francis et al., Intern. J. of Hematol. 68:1-18 (1998); U.S. Pat. No. 4,002,531; U.S. Pat. No. 5,349,052; WO 95/06058; and WO 98/32466, the disclosures of each of which are incorporated herein by reference.

One system for attaching polyethylene glycol directly to amino acid residues of proteins without an intervening linker employs tresylated MPEG, which is produced by the modification of monmethoxy polyethylene glycol (MPEG) using tresylchloride (ClSO2CH2CF3). Upon reaction of protein with tresylated MPEG, polyethylene glycol is directly attached to amine groups of the protein. Thus, the invention includes protein-polyethylene glycol conjugates produced by reacting proteins of the invention with a polyethylene glycol molecule having a 2,2,2-trifluoreothane sulphonyl group.

Polyethylene glycol can also be attached to proteins using a number of different intervening linkers. For example, U.S. Pat. No. 5,612,460, the entire disclosure of which is incorporated herein by reference, discloses urethane linkers for connecting polyethylene glycol to proteins. Protein-polyethylene glycol conjugates wherein the polyethylene glycol is attached to the protein by a linker can also be produced by reaction of proteins with compounds such as MPEG-succinimidylsuccinate, MPEG activated with 1,1′-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate, MPEG-p-nitrophenolcarbonate, and various MPEG-succinate derivatives. A number additional polyethylene glycol derivatives and reaction chemistries for attaching polyethylene glycol to proteins are described in WO 98/32466, the entire disclosure of which is incorporated herein by reference. Pegylated protein products produced using the reaction chemistries set out herein are included within the scope of the invention.

The number of polyethylene glycol moieties attached to each protein of the invention (i.e., the degree of substitution) may also vary. For example, the pegylated proteins of the invention may be linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or more polyethylene glycol molecules. Similarly, the average degree of substitution within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or 18-20 polyethylene glycol moieties per protein molecule. Methods for determining the degree of substitution are discussed, for example, in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).

Antibodies

The present invention further relates to antibodies and T-cell antigen receptors (TCR) which specifically bind the polypeptides of the present invention. The antibodies of the present invention include IgG (including IgG1, IgG2, IgG3, and IgG4), IgA (including IgA1 and IgA2), IgD, IgE, or IgM, and IgY. As used herein, the term “antibody” (Ab) is meant to include whole antibodies, including single-chain whole antibodies, and antigen-binding fragments thereof. Most preferably the antibodies are human antigen binding antibody fragments of the present invention include, but are not limited to, Fab, Fab′ and F(ab′)2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain. The antibodies may be from any animal origin including birds and mammals. Preferably, the antibodies are human, murine, rabbit, goat, guinea pig, camel, horse, or chicken. As used herein, “human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins, as described infra and, for example in, U.S. Pat. No. 5,939,598 by Kucherlapati et al.

Antigen-binding antibody fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entire or partial of the following: hinge region, CH1, CH2, and CH3 domains. Also included in the invention are any combinations of variable region(s) and hinge region, CH1, CH2, and CH3 domains. The present invention further includes monoclonal, polyclonal, chimeric, humanized, and human monoclonal and polyclonal antibodies which specifically bind the polypeptides of the present invention. The present invention further includes antibodies which are anti-idiotypic to the antibodies of the present invention.

The antibodies of the present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for heterologous compositions, such as a heterologous polypeptide or solid support material. See, e.g., WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, A. et al., J. Immunol 147:60-69 (1991); U.S. Pat. Nos. 5,573,920, 4,474,893, 5,601,819, 4,714,681, 4,925,648; Kostelny, S. A. et al, J. Immunol. 148:1547-1553 (1992).

Antibodies of the present invention may be described or specified in terms of the epitope(s) or portion(s) of a polypeptide of the present invention which are recognized or specifically bound by the antibody. The epitope(s) or polypeptide portion(s) may be specified as described herein, e.g., by N-terminal and C-terminal positions, by size in contiguous amino acid residues, or listed in the Tables and Figures. Antibodies which specifically bind any epitope or polypeptide of the present invention may also be excluded. Therefore, the present invention includes antibodies that specifically bind polypeptides of the present invention, and allows for the exclusion of the same.

Antibodies of the present invention may also be described or specified in terms of their cross-reactivity. Antibodies that do not bind any other analog, ortholog, or homolog of the polypeptides of the present invention are included. Antibodies that do not bind polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. Further included in the present invention are antibodies which only bind polypeptides encoded by polynucleotides which hybridize to a polynucleotide of the present invention under stringent hybridization conditions (as described herein). Antibodies of the present invention may also be described or specified in terms of their binding affinity. Preferred binding affinities include those with a dissociation constant or Kd less than 5×10−6M, 10−6M, 5×10−7M, 10−7M, 5×10−8M, 10−8M, 5×10−9M, 10−9M, 5×10−10M, 10−10M, 5×1011M, 10−11M, 5×10−12M, 10−12M, 5×10−13M, 10−13M, 5×10−14M, 10−14M, 5×10−5M, and 10−15M.

The invention also provides antibodies that competitively inhibit binding of an antibody to an epitope of the invention as determined by any method known in the art for determining competitive binding, for example, the immunoassays described herein. In preferred embodiments, the antibody competitively inhibits binding to the epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

Antibodies of the present invention may act as agonists or antagonists of the polypeptides of the present invention. For example, the present invention includes antibodies which disrupt the receptor/ligand interactions with the polypeptides of the invention either partially or fully. The invention features both receptor-specific antibodies and ligand-specific antibodies. The invention also features receptor-specific antibodies which do not prevent ligand binding but prevent receptor activation. Receptor activation (i.e., signaling) may be determined by techniques described herein or otherwise known in the art. For example, receptor activation can be determined by detecting the phosphorylation (e.g., tyrosine or serine/threonine) of the receptor or its substrate by immunoprecipitation followed by western blot analysis (for example, as described supra). In specific embodiments, antibodies are provided that inhibit ligand or receptor activity by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50% of the activity in absence of the antibody.

The invention also features receptor-specific antibodies which both prevent ligand binding and receptor activation as well as antibodies that recognize the receptor-ligand complex, and, preferably, do not specifically recognize the unbound receptor or the unbound ligand. Likewise, included in the invention are neutralizing antibodies which bind the ligand and prevent binding of the ligand to the receptor, as well as antibodies which bind the ligand, thereby preventing receptor activation, but do not prevent the ligand from binding the receptor. Further included in the invention are antibodies which activate the receptor. These antibodies may act as receptor agonists, i.e., potentiate or activate either all or a subset of the biological activities of the ligand-mediated receptor activation. The antibodies may be specified as agonists, antagonists or inverse agonists for biological activities comprising the specific biological activities of the peptides of the invention disclosed herein. The above antibody agonists can be made using methods known in the art. See, e.g., PCT publication WO 96/40281; U.S. Pat. No. 5,811,097; Deng et al, Blood 92(6):1981-1988 (1998); Chen et al, Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol. 161(4):1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214 (1998); Yoon et al, J. Immunol. 160(7):3170-3179 (1998); Prat et al., J. Cell. Sci. 111(Pt2):237-247 (1998); Pitard et al., J. Immunol. Methods205(2): 177-190 (1997); Liautard et al., Cytokine 9(4):233-241 (1997); Carlson et al., J. Biol. Chem. 272(17):11295-11301 (1997); Taryman et al., Neuron 14(4):755-762 (1995); Muller et al., Structure 6(9):1153-1167 (1998); Bartunek et al., Cytokine 8(1):14-20 (1996) (which are all incorporated by reference herein in their entireties).

Antibodies of the present invention have uses that include, but are not limited to, methods known in the art to purify, detect, and target the polypeptides of the present invention including both in vitro and in vivo diagnostic and therapeutic methods. For example, the antibodies have use in immunoassays for qualitatively and quantitatively measuring levels of the polypeptides of the present invention in biological samples. See, e.g., Harlow et al., ANTIBODIES: A LABORATORY MANUAL, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (incorporated by reference in the entirety).

The antibodies of the present invention may be used either alone or in combination with other compositions. The antibodies may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalently and non-covalently conjugations) to polypeptides or other compositions. For example, antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, or toxins. See, e.g., WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 0 396 387.

The antibodies of the invention include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from generating an anti-idiotypic response. For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.

The antibodies of the present invention may be prepared by any suitable method known in the art. Polyclonal antibodies to an antigen-of-interest can be produced by various procedures well known in the art. For example, a polypeptide of the invention can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen. Various adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Such adjuvants are also well known in the art.

Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et alt, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporated by reference in their entireties). The term “monoclonal antibody” is not limited to antibodies produced through hybridoma technology. The term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant and phage display technology.

Hybridoma techniques include those known in the art and taught in Harlow et al, ANTIBODIES: A LABORATORY MANUAL, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); and Hammerling, et al., in: MONOCLONAL ANTIBODIES AND T-CELL HYBRIDOMAS 563-681 (Elsevier, N.Y., 1981) (said references incorporated by reference in their entireties).

Fab and F(ab′)2 fragments may be produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab′)2 fragments).

Alternatively, antibodies of the present invention can be produced through the application of recombinant DNA and phage display technology or through synthetic chemistry using methods known in the art. For example, the antibodies of the present invention can be prepared using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of a phage particle which carries polynucleotide sequences encoding them. Phage with a desired binding property are selected from a repertoire or combinatorial antibody library (e.g. human or murine) by selecting directly with antigen, typically antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and M13 with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman U. et al., J. Immunol. Methods 182:41-50 (1995); Ames, R. S. et al., J. Immunol. Methods 184:177-186 (1995); Kettleborough, C. A. et al., Eur. J. Immunol. 24:952-958 (1994); Persic, L. et al., Gene 187:9-18 (1997); Burton, D. R. et al., Advances in Immunology 57:191-280 (1994); PCT/GB91/01134; WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727 and 5,733,743 (said references incorporated by reference in their entireties).

As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host including mammalian cells, insect cells, plant cells, yeast, and bacteria. For example, techniques to recombinantly produce Fab, Fab′ and F(ab′)2 fragments can also be employed using methods known in the art such as those disclosed in WO 92/22324; Mullinax, R. L. et al., BioTechniques 12:864-869 (1992); and Sawai, H. et al. AJRI 34:26-34 (1995); and Better, M. et al., Science 240: 1041-1043 (1988) (said references incorporated by reference in their entireties).

Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu, L. et al., PNAS 90:7995-7999 (1993); and Skerra, A. et al., Science 240:1038-1040 (1988). For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies. Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies, S. D. et al., J. Immunol. Methods 125:191-202 (1989); and U.S. Pat. No. 5,807,715. Antibodies can be humanized using a variety of techniques including CDR-grafting (EP 0 239 400; WO 91/09967; U.S. Pat. Nos. 5,530,101; and 5,585,089), veneering or resurfacing (EP 0 592 106; EP 0 519 596; Padlan E. A., Molecular Immunology 28(4/5):489-498 (1991); Studnicka G. M. et al., Protein Engineering 7:805-814 (1994); Roguska M. A. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332). Human antibodies can be made by a variety of methods known in the art including phage display methods described above. See also, U.S. Pat. Nos. 4,444,887, 4,716,111, 5,545,806, and 5,814,318; and WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735 and WO 91/10741 (said references incorporated by reference in their entireties).

Further included in the present invention are antibodies recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a polypeptide of the present invention. The antibodies may be specific for antigens other than polypeptides of the present invention. For example, antibodies may be used to target the polypeptides of the present invention to particular cell types, either in vitro or in vivo, by fusing or conjugating the polypeptides of the present invention to antibodies specific for particular cell surface receptors. Antibodies fused or conjugated to the polypeptides of the present invention may also be used in in vitro immunoassays and purification methods using methods known in the art. See e.g., Harbor et al., supra and WO 93/21232; EP 0 439 095; Naramura, M. et al., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies, S. O. et al., PNAS 89:1428-1432 (1992); Fell, H. P. et al., J. Immunol. 146:2446-2452 (1991) (said references incorporated by reference in their entireties).

The present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other than the variable regions. For example, the polypeptides of the present invention may be fused or conjugated to an antibody Fc region, or portion thereof. The antibodyportion fused to a polypeptide of the present invention may comprise the hinge region, CH1 domain, CH2 domain, and CH3 domain or any combination of whole domains or portions thereof. The polypeptides of the present invention may be fused or conjugated to the above antibody portions to increase the in vivo half life of the polypeptides or for use in immunoassays using methods known in the art. The polypeptides may also be fused or conjugated to the above antibody portions to form multimers. For example, Fc portions fused to the polypeptides of the present invention can form dimers through disulfide bonding between the Fc portions. Higher multimeric forms can be made by fusing the polypeptides to portions of IgA and IgM. Methods for fusing or conjugating the polypeptides of the present invention to antibody portions are known in the art. See e.g., U.S. Pat. Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, 5,112,946; EP 0 307 434, EP 0 367 166; WO 96/04388, WO 91/06570; Ashkenazi, A. et al. (1991) PNAS 88:10535-10539; Zheng, X. X. et al. (1995) J. Immunol. 154:5590-5600; and Vil, H. et al. (1992) PNAS 89:11337-11341 (said references incorporated by reference in their entireties).

The invention further relates to antibodies which act as agonists or antagonists of the polypeptides of the present invention. Antibodies which act as agonists or antagonists of the polypeptides of the present invention include, for example, antibodies which disrupt receptor/ligand interactions with the polypeptides of the invention either partially or fully. For example, the present invention includes antibodies which disrupt the ability of the proteins of the invention to multimerize. In another example, the present invention includes antibodies which allow the proteins of the invention to multimerize, but disrupts the ability of the proteins of the invention to bind one or more TR2 receptor(s) or ligand(s) (e.g., AIM II (International Publication No. WO 97/34911), Lymphotoxin-α, and the Herpes virus protein HSV1 gD). In yet another example, the present invention includes antibodies which allow the proteins of the invention to multimerize, and bind TR2 receptor(s) or ligand(s) (e.g., AIM II (International Publication No. WO 97/34911), Lymphotoxin-α, and the Herpes virus protein HSV1 gD), but blocks biological activity associated with the TR2 receptor/ligand complex.

Antibodies which act as agonists or antagonists of the polypeptides of the present invention also include, both receptor-specific antibodies and ligand-specific antibodies. Included are receptor-specific antibodies which do not prevent ligand binding but prevent receptor activation. Receptor activation (i.e., signaling) may be determined by techniques described herein or otherwise known in the art. Also included are receptor-specific antibodies which both prevent ligand binding and receptor activation. Likewise, included are neutralizing antibodies which bind the ligand and prevent binding of the ligand to the receptor, as well as antibodies which bind the ligand, thereby preventing receptor activation, but do not prevent the ligand from binding the receptor. Further included are antibodies which activate the receptor. These antibodies may act as agonists for either all or less than all of the biological activities affected by ligand-mediated receptor activation. The antibodies may be specified as agonists or antagonists for biological activities comprising specific activities disclosed herein. The above antibody agonists can be made using methods known in the art. See e.g., WO 96/40281; U.S. Pat. No. 5,811,097; Deng, B. et al., Blood 92:1981-1988 (1998); Chen, Z. et al., Cancer Res. 58:3668-3678 (1998); Harrop, J. A. et al., J. Immunol 161:1786-1794 (1998); Zhu, Z. et al., Cancer Res. 58:3209-3214 (1998); Yoon, D. Y. et al., J. Immunol 160:3170-3179 (1998); Prat, M. et al., J. Cell. Sci. 111(Pt2):237-247 (1998); Pitard, V. et al., J. Immunol. Methods 205:177-190 (1997); Liautard, J. et al., Cytokine 9(4):233-241 (1997); Carlson, N. G. et al., J. Biol. Chem. 272:11295-11301 (1997); Taryman, R. E. et al., Neuron 14:755-762 (1995); Muller, Y. A. et al., Structure 6:1153-1167 (1998); Bartunek, P. et al., Cytokine 8:14-20 (1996) (said references incorporated by reference in their entireties).

Methods for producing and screening for specific antibodies using hybridoma technology are routine and well-known in the art and are discussed in detail in Example 17. Briefly, mice can be immunized with a polypeptide of the invention or a cell expressing such peptide. Once an immune response is detected, e.g., antibodies specific for the antigen are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well-known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC. Hybridomas are selected and cloned by limited dilution. The hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention. Ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.

Accordingly, the present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell secreting an antibody of the invention wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with an antigen of the invention with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind a polypeptide of the invention.

Antibody fragments that recognize specific epitopes may be generated by known techniques. For example, Fab and F(ab′)2 fragments of the invention may be produced by proteolytic cleavage ofimmunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab′)2 fragments). F(ab′)2 fragments contain the variable region, the light chain constant region and the CH1 domain of the heavy chain.

For example, the antibodies of the present invention can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In a particular, such phage can be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine). Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Phage used in these methods are typically filamentous phage including fd and M13 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al., Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280 (1994); PCT application No. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108; each of which is incorporated herein by reference in its entirety.

As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below. For example, techniques to recombinantly produce Fab, Fab′ and F(ab′)2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869 (1992); and Sawal et al., AJRI 34:26-34 (1995); and Better et al., Science 240:1041-1043 (1988) (said references incorporated by reference in their entireties).

Examples of techniques which can be used to produce single-chain Fvs and antibodies include those described in U.S. Pat. Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040 (1988). For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies. A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, which are incorporated herein by reference in their entireties. Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule. Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which are incorporated herein by reference in their entireties.) Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332).

Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety.

Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. For example, the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring that express human antibodies. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention. Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT publications WO 98/24893; WO 96/34096; WO 96/33735; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; and 5,939,598, which are incorporated by reference herein in their entirety. In addition, companies such as Abgenix, Inc. (Freemont, Calif.) and GenPharm (San Jose, Calif.) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.

Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as “guided selection.” In this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al., Bio/technology 12:899-903 (1988)).

As discussed above, antibodies to the TR2 receptor proteins of the invention can, in turn, be utilized to generate anti-idiotype antibodies that “mimic” TR2 receptors using techniques well known to those skilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444; (1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example, antibodies which bind to TR2 receptors and competitively inhibit TR2 receptor multimerization and/or binding to ligand can be used to generate anti-idiotypes that “mimic” TR2 receptor multimerization and/or binding domain and, as a consequence, bind to and neutralize TR2 receptors and/or their ligand(s). Such neutralizing anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize TR2 receptor ligand(s). For example, such anti-idiotypic antibodies can be used to bind TR2 receptors, or to bind TR2 receptors or ligands, and thereby block TR2 receptor mediated inhibition of apoptosis.

A. Polynucleotides Encoding Antibodies

The invention further provides polynucleotides comprising a nucleotide sequence encoding an antibody of the invention and fragments thereof. The invention also encompasses polynucleotides that hybridize under stringent or lower stringency hybridization conditions, e.g., as defined herein, to polynucleotides that encode an antibody, preferably, that specifically binds to a polypeptide of the invention, preferably, an antibody that binds to a polypeptide having the amino acid sequence of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, or SEQ ID NO:26.

The polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art. For example, if the nucleotide sequence of the antibody is known, a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligation of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably polyA+ RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody of the invention) by PCR amplification using synthetic primers hybridizable to the 3′ and 5′ ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors using any method well known in the art.

Once the nucleotide sequence and corresponding amino acid sequence of the antibody is determined, the nucleotide sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel et al., eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY, which are both incorporated by reference herein in their entireties), to generate antibodies having a different amino acid sequence, for example to create amino acid substitutions, deletions, and/or insertions.

In a specific embodiment, the amino acid sequence of the heavy and/or light chain variable domains may be inspected to identify the sequences of the complementarity determining regions (CDRs) by methods that are well know in the art, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability. Using routine recombinant DNA techniques, one or more of the CDRs may be inserted within framework regions, e.g., into human framework regions to humanize a non-human antibody, as described supra. The framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al., J. Mol Biol 278:457-479 (1998) for a listing of human framework regions). Preferably, the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds a polypeptide of the invention. Preferably, as discussed supra, one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds. Other alterations to the polynucleotide are encompassed by the present invention and within the skill of the art.

In addition, techniques developed for the production of “chimeric antibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used. As described supra, a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region, e.g., humanized antibodies.

Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,694,778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-54) can be adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in E. coli may also be used (Skerra et al., 1988, Science 242:1038-1041).

B. Methods of Producing Antibodies

The antibodies of the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques.

Recombinant expression of an antibody of the invention, or fragment, derivative or analog thereof, e.g., a heavy or light chain of an antibody of the invention, requires construction of an expression vector containing a polynucleotide that encodes the antibody. Once a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof (preferably containing the heavy or light chain variable domain), of the invention has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. The invention, thus, provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a promoter. Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No. 5,122,464) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy or light chain.

The expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention. Thus, the invention includes host cells containing a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, operably linked to a heterologous promoter. In preferred embodiments for the expression of double-chained antibodies, vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to express the antibody molecules of the invention. Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ. These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3 T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter). Preferably, bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., 1986, Gene 45:101; Cockett et al., 1990, Bio/Technology 8:2).

In bacterial systems, a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable. Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem. 24:5503-5509); and the like. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). In general, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix glutathione-agarose beads followed by elution in the presence of free glutathione. The pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The antibody coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts. (e.g. see Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:355-359). Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., 1987, Methods in Enzymol. 153:51-544).

In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used. Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, and in particular, breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell line such as, for example, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stable expression is preferred. For example, cell lines which stably express the antibody molecule may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. This method may advantageously be used to engineer cell lines which express the antibody molecule. Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.

A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 192, Proc. Natl. Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can be employed in tk-, hgprt- or aprt-cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA 77:357; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 Clinical Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIB TECH 11 (5): 155-215); and hygro, which confers resistance to hygromycin (Santerre et al., 1984, Gene 30:147). Methods commonly known in the art of recombinant DNA technology which can be used are described in Ausubel et al. (eds.), 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY; and in Chapters 12 and 13, Dracopoli et al. (eds), 1994, Current Protocols in Human Genetics, John Wiley & Sons, NY.; Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1, which are incorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York, 1987)). When a marker in the vector system expressing antibody is amplifiable, increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et al., 1983, Mol. Cell. Biol. 3:257).

The host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide. The two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides. Alternatively, a single vector may be used which encodes both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, 1986, Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been recombinantly expressed, it may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.

C. Antibody Conjugates

The present invention encompasses antibodies recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a polypeptide (or portion thereof, preferably at least 10, 20 or 50 amino acids of the polypeptide) of the present invention to generate fusion proteins. The fusion does not necessarily need to be direct, but may occur through linker sequences. The antibodies may be specific for antigens other than polypeptides (or portion thereof, preferably at least 10, 20 or 50 amino acids of the polypeptide) of the present invention. For example, antibodies may be used to target the polypeptides of the present invention to particular cell types, either in vitro or in vivo, by fusing or conjugating the polypeptides of the present invention to antibodies specific for particular cell surface receptors. Antibodies fused or conjugated to the polypeptides of the present invention may also be used in in vitro immunoassays and purification methods using methods known in the art. See e.g., Harbor et al., spra, and PCT publication WO 93/21232; EP 439,095; Naramura et al., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies et al., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol. 146:2446-2452 (1991), which are incorporated by reference in their entireties.

The present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other than the variable regions. For example, the polypeptides of the present invention may be fused or conjugated to an antibody Fc region, or portion thereof. The antibody portion fused to a polypeptide of the present invention may comprise the constant region, hinge region, CH1 domain, CH2 domain, and CH3 domain or any combination of whole domains or portions thereof. The polypeptides may also be fused or conjugated to the above antibody portions to form multimers. For example, Fc portions fused to the polypeptides of the present invention can form dimers through disulfide bonding between the Fc portions. Higher multimeric forms can be made by fusing the polypeptides to portions of IgA and IgM. Methods for fusing or conjugating the polypeptides of the present invention to antibody portions are known in the art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCT publications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J. Immunol. 154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA 89:11337-11341 (1992) (said references incorporated by reference in their entireties).

As discussed, supra, the polypeptides of the present invention may be fused or conjugated to the above antibody portions to increase the in vivo half life of the polypeptides or for use in immunoassays using methods known in the art. Further, the polypeptides of the present invention may be fused or conjugated to the above antibody portions to facilitate purification. One reported example describes chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. (EP 394,827; Traunecker et al., Nature 331:84-86 (1988). The polypeptides of the present invention fused or conjugated to an antibody having disulfide-linked dimeric structures (due to the IgG) may also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964 (1995)). In many cases, the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties. (EP A 232,262). Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired. For example, the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations. In drug discovery, for example, human proteins, such as hlL-5 receptor, have been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. (See, D. Bennett et al., J. Molecular Recognition 8:52-58 (1995), K. Johanson et al., J. Biol. Chem. 270:9459-9471 (1995).

Moreover, the antibodies or fragments thereof of the present invention can be fused to marker sequences, such as a peptide to facilitates their purification. In preferred embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the “HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767(1984)) and the “flag” tag.

The present invention further encompasses antibodies or fragments thereof conjugated to a diagnostic or therapeutic agent. The antibodies can be used diagnostically to, for example, monitor the development or progression of a tumor as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. See, for example, U.S. Pat. No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include 125I, 131I, 111In or 99Tc.

Further, an antibody or fragment thereof may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a given biological response, the therapeutic agent or drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, a-interferon, β-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, a thrombotic agent or an anti-angiogenic agent, e.g., angiostatin or endostatin; or, biological response modifiers such as, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophase colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.

Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

Techniques for conjugating such therapeutic moiety to antibodies are well known, see, e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”, Immunol. Rev. 62:119-58 (1982).

Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Pat. No. 4,676,980, which is incorporated herein by reference in its entirety.

An antibody, with or without a therapeutic moiety conjugated to it, administered alone or in combination with cytotoxic factor(s) and/or cytokine(s) can be used as a therapeutic.

D. Assays for Antibody Binding

The antibodies of the invention may be assayed for immunospecific binding by any method known in the art. The immunoassays which can be used include but are not limited to competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is incorporated by reference herein in its entirety). Exemplary immunoassays are described briefly below (but are not intended by way of limitation).

Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g., 1-4 hours) at 4° C., adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 4° C., washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer. The ability of the antibody of interest to immunoprecipitate a particular antigen can be assessed by, e.g., western blot analysis. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the antibody to an antigen and decrease the background (e.g., pre-clearing the cell lysate with sepharose beads). For further discussion regarding immunoprecipitation protocols see, e.g., Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.

Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer (e.g., PB S-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti-human antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32P or 125I) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected and to reduce the background noise. For further discussion regarding western blot protocols see, e.g., Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1.

ELISAs comprise preparing antigen, coating the well of a 96 well microtiter plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen. In ELISAs the antibody of interest does not have to be conjugated to a detectable compound; instead, a second antibody (which recognizes the antibody of interest) conjugated to a detectable compound may be added to the well. Further, instead of coating the well with the antigen, the antibody may be coated to the well. In this case, a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of ELISAs known in the art. For further discussion regarding ELISAs see, e.g., Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1.

The binding affinity of an antibody to an antigen and the off-rate of an antibody-antigen interaction can be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., 3H or 125I) with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen. The affinity of the antibody of interest for a particular antigen and the binding off-rates can be determined from the data by scatchard plot analysis. Competition with a second antibody can also be determined using radioimmunoassays. In this case, the antigen is incubated with antibody of interest is conjugated to a labeled compound (e.g., 3H or 125I) in the presence of increasing amounts of an unlabeled second antibody.

E. Therapeutic Uses

The present invention is further directed to antibody-based therapies which involve administering antibodies of the invention to an animal, preferably a mammal, and most preferably a human, patient for treating one or more of the disclosed diseases, disorders, or conditions. Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (including fragments, analogs and derivatives thereof as described herein) and nucleic acids encoding antibodies of the invention (including fragments, analogs and derivatives thereof and anti-idiotypic antibodies as described herein). The antibodies of the invention can be used to treat, inhibit or prevent diseases, disorders or conditions associated with aberrant expression and/or activity of a polypeptide of the invention, including, but not limited to, autoimmune diseases, disorders, or conditions associated with such diseases or disorders, including, but not limited to, autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenia purpura, autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, relapsing polychondritis, ulcerative colitis, dense deposit disease, rheumatic heart disease, glomerulonephritis (e.g., IgA nephropathy), pemphigus vulgaris, discoid lupus, Multiple Sclerosis, Neuritis, Uveitis Ophthalmia, Polyendocrinopathies, Purpura (e.g., Henloch-Scoenlein purpura), Reiter's Disease, Stiff-Man Syndrome, Autoimmune Pulmonary Inflammation, Guillain-Barre Syndrome, insulin dependent diabetes mellitis, and autoimmune inflammatory eye, autoimmune thyroiditis, hypothyroidism (i.e., Hashimoto's thyroiditis), systemic lupus erhythematosus, Goodpasture's syndrome, Pemphigus, Receptor autoimmunities such as, for example, (a) Graves' Disease, (b) Myasthenia Gravis, and (c) insulin resistance, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, rheumatoid arthritis, schleroderma with anti-collagen antibodies, mixed connective tissue disease, polymyositis/dermatomyositis, pernicious anemia, idiopathic Addison's disease, infertility, glomerulonephritis such as primary glomerulonephritis and IgA nephropathy, bullous pemphigoid, Sjogren's syndrome, diabetes millitus, and adrenergic drug resistance (including adrenergic drug resistance with asthma or cystic fibrosis), chronic active hepatitis, primary biliary cirrhosis, other endocrine gland failure, vitiligo, vasculitis, post-MI, cardiotomy syndrome, urticaria, atopic dermatitis, asthma, inflammatory myopathies, graft v. host diseases (GVHD) and other inflammatory, granulamatous, degenerative, and atrophic disorders).

In a specific embodiment, antibodies of the invention are be used to treat, inhibit, prognose, diagnose or prevent rheumatoid arthritis.

In another specific embodiment, antibodies of the invention are used to treat, inhibit, prognose, diagnose or prevent systemic lupus erythematosis.

Additionally, the antibodies of the invention can be used to treat, inhibit or prevent diseases, disorders or conditions associated with immunodeficiencies including, but not limited to, severe combined immunodeficiency (SCID)-X linked, SCID-autosomal, adenosine deaminase deficiency (ADA deficiency), X-linked agammaglobulinemia (XLA), Bruton's disease, congenital agammaglobulinemia, X-linked infantile agammaglobulinemia, acquired agammaglobulinemia, adult onset agammaglobulinemia, late-onset agammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia, transient hypogammaglobulinemia of infancy, unspecified hypogammaglobulinemia, agammaglobulinemia, common variable immunodeficiency (CVID) (acquired), Wiskott-Aldrich Syndrome (WAS), X-linked immunodeficiency with hyper IgM, non X-linked immunodeficiency with hyper IgM, selective IgA deficiency, IgG subclass deficiency (with or without IgA deficiency), antibody deficiency with normal or elevated Igs, immunodeficiency with thymoma, Ig heavy chain deletions, kappa chain deficiency, B cell lymphoproliferative disorder (BLPD), selective IgM immunodeficiency, recessive agammaglobulinemia (Swiss type), reticular dysgenesis, neonatal neutropenia, autoimmune neutropenia, severe congenital leukopenia, thymic alymphoplasia-aplasia or dysplasia with immunodeficiency, ataxia-telangiectasia, short limbed dwarfism, X-linked lymphoproliferative syndrome (XLP), Nezelof syndrome-combined immunodeficiency with Igs, purine nucleoside phosphorylase deficiency (PNP), MHC Class II deficiency (Bare Lymphocyte Syndrome) and severe combined immunodeficiency.

Antibodies of the invention are used to prevent graft rejection and inflammation and for the treatment of arthritis.

The treatment and/or prevention of diseases and disorders associated with aberrant expression and/or activity of a polypeptide of the invention includes, but is not limited to, alleviating symptoms associated with those diseases and disorders. Antibodies of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.

A summary of the ways in which the antibodies of the present invention may be used therapeutically includes binding polynucleotides or polypeptides of the present invention locally or systemically in the body or by direct cytotoxicity of the antibody, e.g., as mediated by complement (CDC) or by effector cells (ADCC). Some of these approaches are described in more detail below. Armed with the teachings provided herein, one of ordinary skill in the art will know how to use the antibodies of the present invention for diagnostic, monitoring or therapeutic purposes without undue experimentation.

The antibodies of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3 and IL-7), for example, which serve to increase the number or activity of effector cells which interact with the antibodies.

The antibodies of the invention may be administered alone or in combination with other types of treatments (e.g., radiation therapy, chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents). Generally, administration of products of a species origin or species reactivity (in the case of antibodies) that is the same species as that of the patient is preferred. Thus, in a preferred embodiment, human antibodies, fragments derivatives, analogs, or nucleic acids, are administered to a human patient for therapy or prophylaxis.

It is preferred to use high affinity and/or potent in vivo inhibiting and/or neutralizing antibodies against polypeptides or polynucleotides of the present invention, fragments or regions thereof, for both immunoassays directed to and therapy of disorders related to polynucleotides or polypeptides, including fragments thereof, of the present invention. Such antibodies, fragments, or regions, will preferably have an affinity for polynucleotides or polypeptides, including fragments thereof. Preferred binding affinities include those with a dissociation constant or Kd less than 5×10−6M, 10−6M, 5×10−7M, 10−7M, 5×10−8M, 10−8M, 5×10−9M, 10−9M, 5×10−10M, 10−10M, 5×10−11M, 10−11M, 5×10−12M, 10−12M, 5×10−13M, 10−13M, 5×10−14M, 10×−14M, 5×10−5M, and 10−15M.

Transgenic Non-Human Animals

The proteins of the invention can also be expressed in transgenic animals. Animals of any species, including, but not limited to, mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep, cows and non-human primates, e.g., baboons, monkeys, and chimpanzees may be used to generate transgenic animals. In a specific embodiment, techniques described herein or otherwise known in the art, are used to express polypeptides of the invention in humans, as part of a gene therapy protocol.

Any technique known in the art may be used to introduce the transgene (i.e., nucleic acids of the invention) into animals to produce the founder lines of transgenic animals. Such techniques include, but are not limited to, pronuclear microinjection (Paterson et al., Appl. Microbiol. Biotechnol. 40:691-698 (1994); Carver et al., Biotechnology (NY) 11:1263-1270 (1993); Wright et al., Biotechnology (NY) 9:830-834 (1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989)); retrovirus mediated gene transfer into germ lines (Van der Putten et al., Proc. Natl. Acad. Sci., USA 82:6148-6152 (1985)), blastocysts or embryos; gene targeting in embryonic stem cells (Thompson et al., Cell 56:313-321 (1989)); electroporation of cells or embryos (Lo, Mol. Cell. Biol. 3:1803-1814 (1983)); introduction of the polynucleotides of the invention using a gene gun (see, e.g., Ulmer et al., Science 259:1745 (1993); introducing nucleic acid constructs into embryonic pleuripotent stem cells and transferring the stem cells back into the blastocyst; and sperm-mediated gene transfer (Lavitrano et al., Cell 57:717-723 (1989); etc. For a review of such techniques, see Gordon, “Transgenic Animals,” Intl. Rev. Cytol. 115:171-229 (1989), which is incorporated by reference herein in its entirety. See, also, U.S. Pat. No. 5,464,764 (Capecchi, et al., Positive-Negative Selection Methods and Vectors); U.S. Pat. No. 5,631,153 (Capecchi, et al., Cells and Non-Human Organisms Containing Predetermined Genomic Modifications and Positive-Negative Selection Methods and Vectors for Making Same); U.S. Pat. No. 4,736,866 (Leder, et al., Transgenic Non-Human Animals); and U.S. Pat. No. 4,873,191 (Wagner, et al., Genetic Transformation of Zygotes); each of which is hereby incorporated by reference in its entirety. Further, the contents of each of the documents recited in this paragraph is herein incorporated by reference in its entirety.

Any technique known in the art may be used to produce transgenic clones containing polynucleotides of the invention, for example, nuclear transfer into enucleated oocytes of nuclei from cultured embryonic, fetal, or adult cells induced to quiescence (Campell et al., Nature 380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)), each of which is herein incorporated by reference in its entirety).

The present invention provides for transgenic animals that carry the transgene in all their cells, as well as animals which carry the transgene in some, but not all their cells, i.e., mosaic animals or chimeric animals. The transgene may be integrated as a single transgene or as multiple copies such as in concatamers, e.g., head-to-head tandems or head-to-tail tandems. The transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA 89:6232-6236 (1992)). The regulatory sequences required for such a cell-type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art. When it is desired that the polynucleotide transgene be integrated into the chromosomal site of the endogenous gene, gene targeting is preferred. Briefly, when such a technique is to be utilized, vectors containing some nucleotide sequences homologous to the endogenous gene are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous gene. The transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous gene in only that cell type, by following, for example, the teaching of Gu et al. (Gu et al., Science 265:103-106(1994)). The regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art. The contents of each of the documents recited in this paragraph is herein incorporated by reference in its entirety.

Once transgenic animals have been generated, the expression of the recombinant gene may be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to verify that integration of the transgene has taken place. The level of mRNA expression of the transgene in the tissues of the transgenic animals may also be assessed using techniques which include, but are not limited to, Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and reverse transcriptase-PCR (rt-PCR). Samples of transgenic gene-expressing tissue may also be evaluated immunocytochemically or immunohistochemically using antibodies specific for the transgene product.

Once the founder animals are produced, they may be bred, inbred, outbred, or crossbred to produce colonies of the particular animal. Examples of such breeding strategies include, but are not limited to: outbreeding of founder animals with more than one integration site in order to establish separate lines; inbreeding of separate lines in order to produce compound transgenics that express the transgene at higher levels because of the effects of additive expression of each transgene; crossing of heterozygous transgenic animals to produce animals homozygous for a given integration site in order to both augment expression and eliminate the need for screening of animals by DNA analysis; crossing of separate homozygous lines to produce compound heterozygous or homozygous lines; and breeding to place the transgene on a distinct background that is appropriate for an experimental model of interest.

Transgenic and “knock-out” animals of the invention have uses which include, but are not limited to, animal model systems useful in elaborating the biological function of TR2 receptor polypeptides, studying conditions and/or disorders associated with aberrant TR2 receptor expression, and in screening for compounds effective in ameliorating such conditions and/or disorders.

In further embodiments of the invention, cells that are genetically engineered to express the proteins of the invention, or alternatively, that are genetically engineered not to express the proteins of the invention (e.g., knockouts) are administered to a patient in vivo. Such cells may be obtained from the patient (i.e., animal, including human) or an MHC compatible donor and can include, but are not limited to fibroblasts, bone marrow cells, blood cells (e.g., lymphocytes), adipocytes, muscle cells, endothelial cells, etc. The cells are genetically engineered in vitro using recombinant DNA techniques to introduce the coding sequence of polypeptides of the invention into the cells, or alternatively, to disrupt the coding sequence and/or endogenous regulatory sequence associated with the polypeptides of the invention, e.g., by transduction (using viral vectors, and preferably vectors that integrate the transgene into the cell genome) or transfection procedures, including, but not limited to, the use of plasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. The coding sequence of the polypeptides of the invention can be placed under the control of a strong constitutive or inducible promoter or promoter/enhancer to achieve expression, and preferably secretion, of the polypeptides of the invention. The engineered cells which express and preferably secrete the polypeptides of the invention can be introduced into the patient systemically, e.g., in the circulation, or intraperitoneally. Alternatively, the cells can be incorporated into a matrix and implanted in the body, e.g., genetically engineered fibroblasts can be implanted as part of a skin graft; genetically engineered endothelial cells can be implanted as part of a lymphatic or vascular graft. (See, e.g., Anderson et al. U.S. Pat. No. 5,399,349; and Mulligan & Wilson, U.S. Pat. No. 5,460,959, each of which is incorporated by reference herein in its entirety).

When the cells to be administered are non-autologous or non-MHC compatible cells, they can be administered using well known techniques which prevent the development of a host immune response against the introduced cells. For example, the cells may be introduced in an encapsulated form which, while allowing for an exchange of components with the immediate extracellular environment, does not allow the introduced cells to be recognized by the host immune system.

Detection of Disease States

The TNF-family ligands induce various cellular responses by binding to TNF-family receptors, including the TR2 receptors of the present invention. TNF-β, a potent ligand of the TNF receptor proteins, is known to be involved in a number of biological processes including lymphocyte development, tumor necrosis, induction of an antiviral state, activation of polymorphonuclear leukocytes, induction of class I major histocompatibility complex antigens on endothelial cells, induction of adhesion molecules on endothelium and growth hormone stimulation (Ruddle and Horner, Prog. Allergy, 40:162-182 (1988)). TNF-α, also a ligand of the TNF receptor proteins, has been reported to have a role in the rapid necrosis of tumors, immunostimulation, autoimmune disease, graft rejection, producing an anti-viral response, septic shock, cerebral malaria, cytotoxicity, protection against deleterious effects of ionizing radiation produced during a course of chemotherapy, such as denaturation of enzymes, lipid peroxidation and DNA damage (Nata et al., J. Immunol. 136(7):2483 (1987); Porter, Tibtech 9:158-162 (1991)), growth regulation, vascular endothelium effects and metabolic effects. TNF-α also triggers endothelial cells to secrete various factors, including PAI-1, IL-1, GM-CSF and IL-6 to promote cell proliferation. In addition, TNF-α up-regulates various cell adhesion molecules such as E-Selectin, ICAM-1 and VCAM-1. TNF-α and the Fas ligand have also been shown to induce programmed cell death.

Cells which express the TR2 polypeptides and are believed to have a potent cellular response to TR2 receptor ligands include B lymphocytes (CD19+), both CD4+ and CD8+ T lymphocytes, monocytes, endothelial cells and other cell types shown in Tables V and VI. By “a cellular response to a TNF-family ligand” is intended any genotypic, phenotypic, and/or morphologic change to a cell, cell line, tissue, tissue culture or patient that is induced by a TNF-family ligand. As indicated, such cellular responses include not only normal physiological responses to TNF-family ligands, but also diseases associated with increased cell proliferation or the inhibition of increased cell proliferation, such as by the inhibition of apoptosis. Apoptosis-programmed cell death-is a physiological mechanism involved in the deletion of peripheral T lymphocytes of the immune system, and its dysregulation can lead to a number of different pathogenic processes (Ameisen, J. C., AIDS 8: 1197-1213 (1994); Krammer, P. H. et al., Curr. Opin. Immunol. 6:279-289 (1994)).

It is believed that certain tissues in mammals with specific disease states associated with aberrant cell survival express significantly altered levels of TR2 receptor protein and mRNA encoding TR2 receptor protein when compared to a corresponding “standard” mammal, i.e., a mammal of the same species not having the disease state. Further, since some forms of this protein are secreted, it is believed that enhanced levels of TR2 receptor protein can be detected in certain body fluids (e.g., sera, plasma, urine, and spinal fluid) from mammals with the disease state when compared to sera from mammals of the same species not having the disease state. Thus, the invention provides a diagnostic method useful during diagnosis of disease states, which involves assaying the expression level of the gene encoding TR2 receptor protein in mammalian cells or body fluid and comparing the gene expression level with a standard TR2 receptor gene expression level, whereby an increase or decrease in the gene expression level over the standard is indicative of certain disease states associated with aberrant cell survival.

Where diagnosis of a disease state involving the TR2 receptors of the present invention has already been made according to conventional methods, the present invention is useful as a prognostic indicator, whereby patients exhibiting significantly aberrant TR2 receptor gene expression will experience a worse clinical outcome relative to patients expressing the gene at a lower level.

By “assaying the expression level of the gene encoding TR2 receptor protein” is intended qualitatively or quantitatively measuring or estimating the level of TR2, TR2-SV1 and/or TR2-SV2 receptor protein or the level of the mRNA encoding TR2, TR2-SV1 and/or TR2-SV2 receptor protein in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g., by comparing to TR2, TR2-SV1 and/or TR2-SV2 receptor protein level or mRNA level in a second biological sample).

Preferably, TR2 receptor protein level or mRNA level in the first biological sample is measured or estimated and compared to a standard TR2 receptor protein level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the disease state. As will be appreciated in the art, once a standard TR2 receptor protein level or mRNA level is known, it can be used repeatedly as a standard for comparison.

By “biological sample” is intended any biological sample obtained from an individual, cell line, tissue culture, or other source which contains TR2 receptor protein or mRNA. Biological samples include mammalian body fluids (such as sera, plasma, urine, synovial fluid and spinal fluid) which contain secreted mature TR2 receptor protein, and thymus, prostate, heart, placenta, muscle, liver, spleen, lung, kidney and other tissues. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. Where the biological sample is to include mRNA, a tissue biopsy is the preferred source.

Diseases associated with increased cell survival, or the inhibition of apoptosis, include cancers (such as follicular lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors, including, but not limited to, colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer); autoimmune disorders (such as systemic lupus erythematosus and immune-related glomerulonephritis rheumatoid arthritis) and viral infections (such as Herpes viruses, pox viruses and adenoviruses), information graft v. host disease, acute graft rejection, and chronic graft rejection. Diseases associated with decreased cell survival, or increased apoptosis, include AIDS; neurodegenerative disorders (such as Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration); myelodysplastic syndromes (such as aplastic anemia), ischemic injury (such as that caused by myocardial infarction, stroke and reperfusion injury), toxin-induced liver disease (such as that caused by alcohol), septic shock, cachexia and anorexia.

In preferred embodiments TR2, TR2-SV1 and/or TR2-SV2 polynucleotides or polypeptides of the invention are used to treat or prevent autoimmune diseases and/or inhibit the growth, progression, and/or metastasis of cancers, including, but not limited to, those cancers disclosed herein, such as, for example, lymphocytic leukemias (including, for example, MLL and chronic lymphocytic leukemia (CLL)) and follicular lymphomas. In another embodiment TR2, TR2-SV1 and/or TR2-SV2 polynucleotides or polypeptides of the invention are used to activate, differentiate or proliferate cancerous cells or tissue (e.g., B cell lineage related cancers (e.g., CLL and MLL), lymphocytic leukemia, or lymphoma) and thereby render the cells more vulnerable to cancer therapy (e.g., chemotherapy or radiation therapy).

Assays available to detect levels of soluble receptors are well known to those of skill in the art, for example, radioimmunoassays, competitive-binding assays, Western blot analysis, and preferably an ELISA assay may be employed.

TR2 receptor-protein specific antibodies can be raised against intact TR2 receptor protein or an antigenic polypeptide fragment thereof, which may presented together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse) or, if it is long enough (at least about 25 amino acids), without a carrier.

As used herein, the term “antibody” (Ab) or “monoclonal antibody” (mAb) is meant to include intact molecules as well as antibody fragments (such as, for example, Fab and F(ab′)2 fragments) which are capable of specifically binding to TR2 receptor protein. Fab and F(ab′)2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). Thus, these fragments are preferred.

The antibodies of the present invention may be prepared by any of a variety of methods. For example, cells expressing TR2, TR2-SV1 and/or TR2-SV2 receptor protein or an antigenic fragment thereof can be administered to an animal in order to induce the production of sera containing polyclonal antibodies. In a preferred method, a preparation of TR2, TR2-SV1 and/or TR2-SV2 receptor protein is prepared and purified to render it substantially free of natural contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity.

In the most preferred method, the antibodies of the present invention are monoclonal antibodies (or TR2 receptor protein binding fragments thereof). Such monoclonal antibodies can be prepared using hybridoma technology (Kohler et al., Nature 256:495 (1975); Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler et al., Eur. J. Immunol. 6:292 (1976); Hammerling et al., In: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., (1981) pp. 563-681). In general, such procedures involve immunizing an animal (preferably a mouse) with a TR2 receptor protein antigen or, more preferably, with a TR2 receptor protein-expressing cell. Suitable cells can be recognized by their capacity to bind anti-TR2 receptor protein antibody. Such cells may be cultured in any suitable tissue culture medium; however, it is preferable to culture cells in Earle's modified Eagle's medium supplemented with 10% fetal bovine serum (inactivated at about 56° C.), and supplemented with about 10 g/l of nonessential amino acids, about 1,000 U/ml of penicillin, and about 100 μg/ml of streptomycin. The splenocytes of such mice are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line may be employed in accordance with the present invention; however, it is preferable to employ the parent myeloma cell line (SP2O), available from the American Type Culture Collection, Rockville, Md. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et al. (Gastroenterology 80:225-232 (1981)). The hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the TR2 receptor protein antigen.

Agonists and Antagonists of TR2 Receptor Function

In one aspect, the present invention is directed to a method for inhibiting a TR2 activity induced by a TNF-family ligand (e.g., cell proliferation, hematopoietic development), which involves administering to a cell which expresses a TR2 polypeptide an effective amount of a TR2 receptor ligand, analog or an antagonist capable of decreasing TR2 receptor mediated signaling. Preferably, TR2 receptor mediated signaling is increased to treat a disease wherein increased cell proliferation is exhibited. An antagonist can include soluble forms of the TR2 receptors and antibodies directed against the TR2 polypeptides which block TR2 receptor mediated signaling. Preferably, TR2 receptor mediated signaling is decreased to treat a disease.

In a further aspect, the present invention is directed to a method for increasing cell proliferation induced by a TNF-family ligand, which involves administering to a cell which expresses a TR2 polypeptide an effective amount of an agonist capable of increasing TR2 receptor mediated signaling. Preferably, TR2 receptor mediated signaling is increased to treat a disease wherein decreased cell proliferation is exhibited. Agonists of the present invention include monoclonal antibodies directed against the TR2 polypeptides which stimulate TR2 receptor mediated signaling. Preferably, TR2 receptor mediated signaling is increased to treat a disease.

By “agonist” is intended naturally occurring and synthetic compounds capable of enhancing cell proliferation and differentiation mediated by TR2 polypeptides. Such agonists include agents which increase expression of TR2 receptors or increase the sensitivity of the expressed receptor. By “antagonist” is intended naturally occurring and synthetic compounds capable of inhibiting TR2 mediated cell proliferation and differentiation. Such antagonists include agents which decrease expression of TR2 receptors or decrease the sensitivity of the expressed receptor. Whether any candidate “agonist” or “antagonist” of the present invention can enhance or inhibit cell proliferation and differentiation can be determined using art-known TNF-family ligand/receptor cellular response assays, including those described in more detail below.

One such screening technique involves the use of cells which express the receptor (for example, transfected CHO cells) in a system which measures extracellular pH changes caused by receptor activation, for example, as described in Science 246:181-296 (October 1989). For example, compounds may be contacted with a cell which expresses the receptor polypeptide of the present invention and a second messenger response, e.g., signal transduction or pH changes, may be measured to determine whether the potential compound activates or inhibits the receptor.

Another such screening technique involves introducing RNA encoding the receptor into Xenopus oocytes to transiently express the receptor. The receptor oocytes may then be contacted with the receptor ligand and a compound to be screened, followed by detection of inhibition or activation of a calcium signal in the case of screening for compounds which are thought to inhibit activation of the receptor.

Another method involves screening for compounds which inhibit activation of the receptor polypeptide of the present invention antagonists by determining inhibition of binding of labeled ligand to cells which have the receptor on the surface thereof. Such a method involves transfecting a eukaryotic cell with DNA encoding the receptor such that the cell expresses the receptor on its surface and contacting the cell with a compound in the presence of a labeled form of a known ligand. The ligand can be labeled, e.g., by radioactivity. The amount of labeled ligand bound to the receptors is measured, e.g., by measuring radioactivity of the receptors. If the compound binds to the receptor as determined by a reduction of labeled ligand which binds to the receptors, the binding of labeled ligand to the receptor is inhibited.

Soluble forms of the polypeptides of the present invention may be utilized in the ligand binding assay described above. These forms of the TR2 receptors are contacted with ligands in the extracellular medium after they are secreted. A determination is then made as to whether the secreted protein will bind to TR2 receptor ligands.

Further screening assays for agonist and antagonist of the present invention are described in Tartaglia, L. A., and Goeddel, D. V., J. Biol. Chem. 267(7):4304-4307 (1992).

Thus, in a further aspect, a screening method is provided for determining whether a candidate agonist or antagonist is capable of enhancing or inhibiting a cellular response to a TNF-family ligand. The method involves contacting cells which express TR2 polypeptides with a candidate compound and a TNF-family ligand, assaying a cellular response, and comparing the cellular response to a standard cellular response, the standard being assayed when contact is made with the ligand in absence of the candidate compound, whereby an increased cellular response over the standard indicates that the candidate compound is an agonist of the ligand/receptor signaling pathway and a decreased cellular response compared to the standard indicates that the candidate compound is an antagonist of the ligand/receptor signaling pathway. By “assaying a cellular response” is intended qualitatively or quantitatively measuring a cellular response to a candidate compound and/or a TNF-family ligand (e.g., determining or estimating an increase or decrease in T cell proliferation or tritiated thymidine labeling). By the invention, a cell expressing a TR2 polypeptide can be contacted with either an endogenous or exogenously administered TNF-family ligand.

In an additional aspect, a thymocyte proliferation assay may be employed to identify both ligands and potential drug candidates. For example, thymus cells are disaggregated from tissue and grown in culture medium. Incorporation of DNA precursors such as 3H-thymidine or 5-bromo-2′-deoxyuridine (BrdU) is monitored as a parameter for DNA synthesis and cellular proliferation. Cells which have incorporated BrdU into DNA can be detected using a monoclonal antibody against BrdU and measured by an enzyme or fluorochrome-conjugated second antibody. The reaction is quantitated by fluorimetry or by spectrophotometry. Two control wells and an experimental well are set up as above and TNF-β or cognate ligand is added to all wells while soluble receptor polypeptides of the present invention are added individually to the second control wells, with the experimental well containing a compound to be screened. The ability of the compound to be screened to stimulate or inhibit the above interaction may then be quantified.

Agonists according to the present invention include compounds such as, for example, TNF-family ligand peptide fragments, transforming growth factor β, and neurotransmitters (such as glutamate, dopamine, N-methyl-D-aspartate). Preferred agonist include polyclonal and monoclonal antibodies raised against a TR2 polypeptide, or a fragment thereof. Such agonist antibodies raised against a TNF-family receptor are disclosed in Tartaglia, L. A., et al., Proc. Natl. Acad. Sci. USA 88:9292-9296 (1991); and Tartaglia, L. A., and Goeddel, D. V., J. Biol. Chem. 267 (7):4304-4307 (1992). See, also, PCT Application WO 94/09137. Further preferred agonists include chemotherapeutic drugs such as, for example, cisplatin, doxorubicin, bleomycin, cytosine arabinoside, nitrogen mustard, methotrexate and vincristine. Others include ethanol and β-amyloid peptide. (Science 267:1457-1458 (1995)).

Antagonist according to the present invention include soluble forms of the TR2 receptors (e.g., fragments of the TR2 receptor shown in FIG. 1A-1B that include the ligand binding domain from the extracellular region of the full length receptor). Such soluble forms of the receptor, which may be naturally occurring or synthetic, antagonize TR2, TR2-SV1 or TR2-SV2 mediated signaling by competing with the cell surface bound forms of the receptor for binding to TNF-family ligands. Antagonists of the present invention also include antibodies specific for TNF-family ligands and TR2-Fc fusion proteins such as the one described below in Examples 5 and 6.

By a “TNF-family ligand” is intended naturally occurring, recombinant, and synthetic ligands that are capable of binding to a member of the TNF receptor family and inducing the ligand/receptor signaling pathway. Members of the TNF ligand family include, but are not limited to, TNF-α, lymphotoxin-α (LT-α, also known as TNF-β), LT-β (found in complex heterotrimer LT-α2-β), FasL, CD40L, CD27L, CD30L, 4-lBBL, OX40L and nerve growth factor (NGF).

The experiments set forth in Example 6 demonstrate that the TR2 receptors of the present invention are capable of inducing the proliferation of lymphocytes. Further, such proliferation can be inhibited by a TR2 protein fragment fused to an Fc antibody fragment. Thus, specifically included within the scope of the invention are TR2 receptor/Fc fusion proteins, and nucleic acid molecules which encode such proteins. These fusion proteins include those having amino acid sequences of the extracellular domains of the TR2 proteins of the invention. Examples of portions of TR2 extracellular domains which are useful in the preparation of TR2 receptor/Fc fusion proteins include amino acids 1 to 192, 37 to 192, 50 to 192 and 100 to 192 in SEQ ID NO:2.

TNFα has been shown to protect mice from infection with Herpes simplex virus type 1 (HSV-1). Rossol-Voth, R. et al., J. Gen. Virol. 72:143-147 (1991). The mechanism of the protective effect of TNFα is unknown but appears to involve neither interferons not NK cell killing. One member of the TNFR family has been shown to mediate HSV-1 entry into cells. Montgomery, R. et al., Eur. Cytokine Newt. 7:159 (1996). Further, antibodies specific for the extracellular domain of this TNFR block HSV-1 entry into cells. Thus, TR2 receptors of the present invention include both TR2 amino acid sequences and antibodies capable of preventing TNFR mediated viral entry into cells. Such sequences and antibodies can function by either competing with cell surface localized TNFR for binding to virus or by directly blocking binding of virus to cell surface receptors.

Similarly, antibodies specific for the extracellular domain of the TR2 receptors of the invention, as well as other TR2 antagonists, can also block HSV-1 entry into cells. These antagonists are thus useful in the treatment and prevention of Herpes simplex infections.

Antibodies according to the present invention may be prepared by any of a variety of methods using TR2 receptor immunogens of the present invention. Such TR2 receptor immunogens include the TR2 receptor protein shown in SEQ ID NO:26, FIG. 1A-1B (SEQ ID NO:2) and the TR2-SV1 (FIG. 4A-4B (SEQ ID NO:5)) and TR2-SV2 (FIG. 7A-7B (SEQ ID NO:8)) polypeptides (any of which may or may not include a leader sequence) and polypeptide fragments of the receptors comprising, or alternatively consisting of, the ligand binding, extracellular, transmembrane, the intracellular domains of the TR2 receptors, or any combination thereof.

Polyclonal and monoclonal antibody agonist or antagonist according to the present invention can be raised according to the methods disclosed in Tartaglia and Goeddel, J. Biol. Chem. 267(7):4304-4307 (1992)); Tartaglia et al., Cell 73:213-216 (1993)), and PCT Application WO 94/09137. The term “antibody” (Ab) or “monoclonal antibody” (mAb) as used herein is meant to include intact molecules as well as fragments thereof (such as, for example, Fab and F(ab′)2 fragments) which are capable of binding an antigen. Fab and F(ab′)2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody (Wahl et al., J. Nucl. Med 24:316-325 (1983)).

In a preferred method, antibodies according to the present invention are mAbs. Such mAbs can be prepared using hybridoma technology (Kohler and Millstein, Nature 256:495-497 (1975) and U.S. Pat. No. 4,376,110; Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988; Monoclonal Antibodies and Hybridomas: A New Dimension in Biological Analyses, Plenum Press, New York, N.Y., 1980; Campbell, “Monoclonal Antibody Technology,” In: Laboratory Techniques in Biochemistry and Molecular Biology, Volume 13 (Burdon et al., eds.), Elsevier, Amsterdam (1984)).

Proteins and other compounds which bind the TR2 receptor domains are also candidate agonist and antagonist according to the present invention. Such binding compounds can be “captured” using the yeast two-hybrid system (Fields and Song, Nature 340:245-246 (1989)). A modified version of the yeast two-hybrid system has been described by Roger Brent and his colleagues (Gyuris, J. et al., Cell 75:791-803 (1993); Zervos, A. S. et al., Cell 72:223-232 (1993)). Preferably, the yeast two-hybrid system is used according to the present invention to capture compounds which bind to the ligand binding, extracellular, intracellular, and transmembrane domains of the TR2 receptors. Such compounds are good candidate agonist and antagonist of the present invention.

Using the two-hybrid assay described above, the intracellular domain of the TR2 receptor, or a portion thereof, may be used to identify cellular proteins which interact with the receptor in vivo. Such an assay may also be used to identify ligands with potential agonistic or antagonistic activity of TR2 receptor function. This screening assay has previously been used to identify protein which interact with the cytoplasmic domain of the murine TNF-RII and led to the identification of two receptor associated proteins. Rothe, M. et al., Cell 78:681 (1994). Such proteins and amino acid sequences which bind to the cytoplasmic domain of the TR2 receptors are good candidate agonist and antagonist of the present invention.

Other screening techniques include the use of cells which express the polypeptide of the present invention (for example, transfected CHO cells) in a system which measures extracellular pH changes caused by receptor activation, for example, as described in Science, 246:181-296 (1989). In another example, potential agonists or antagonists may be contacted with a cell which expresses the polypeptide of the present invention and a second messenger response, e.g., signal transduction may be measured to determine whether the potential antagonist or agonist is effective.

The TR2 receptor agonists may be employed to stimulate ligand activities, such as inhibition of tumor growth and necrosis of certain transplantable tumors. The agonists may also be employed to stimulate cellular differentiation, for example, T-cell, fibroblasts and hemopoietic cell differentiation. Agonists to the TR2 receptor may also augment TR2's role in the host's defense against microorganisms and prevent related diseases (infections such as that from Listeria monocytogenes) and Chlamidiae. The agonists may also be employed to protect against the deleterious effects of ionizing radiation produced during a course of radiotherapy, such as denaturation of enzymes, lipid peroxidation, and DNA damage.

Agonists to the receptor polypeptides of the present invention may be used to augment TNF's role in host defenses against microorganisms and prevent related diseases. The agonists may also be employed to protect against the deleterious effects of ionizing radiation produced during a course of radiotherapy, such as denaturation of enzymes, lipid peroxidation, and DNA damage.

The agonists may also be employed to mediate an anti-viral response, to regulate growth, to mediate the immune response and to treat immunodeficiencies related to diseases such as HIV by increasing the rate of lymphocyte proliferation and differentiation.

The antagonists to the polypeptides of the present invention may be employed to inhibit ligand activities, such as stimulation of tumor growth and necrosis of certain transplantable tumors. The antagonists may also be employed to inhibit cellular differentiation, for example, T-cell, fibroblasts and hemopoietic cell differentiation. Antagonists may also be employed to treat autoimmune diseases, for example, graft versus host rejection and allograft rejection, and T-cell mediated autoimmune diseases such as AIDS. It has been shown that T-cell proliferation is stimulated via a type 2 TNF receptor. Accordingly, antagonizing the receptor may prevent the proliferation of T-cells and treat T-cell mediated autoimmune diseases.

The state of immunodeficiency that defines AIDS is secondary to a decrease in the number and function of CD4+ T-lymphocytes. Recent reports estimate the daily loss of CD4+ T cells to be between 3.5×107 and 2×109 cells (Wei X., et al., Nature 373:117-122 (1995)). One cause of CD4+ T cell depletion in the setting of HIV infection is believed to be HIV-induced apoptosis. Indeed, HIV-induced apoptotic cell death has been demonstrated not only in vitro but also, more importantly, in infected individuals (Ameisen, J. C., AIDS 8: 1197-1213 (1994); Finkel, T. H., and Banda, N. K., Curr. Opin. Immunol. 6:605-615 (1995); Muro-Cacho, C. A. et al., J. Immunol. 154:5555-5566 (1995)). Furthermore, apoptosis and CD4+ T-lymphocyte depletion is tightly correlated in different animal models of AIDS (Brunner, T., et al., Nature 373:441-444 (1995); Gougeon, M. L., et al., AIDS Res. Hum. Retroviruses 9:553-563 (1993)) and, apoptosis is not observed in those animal models in which viral replication does not result in AIDS (Gougeon, M. L. et al., AIDS Res. Hum. Retroviruses 9:553-563 (1993)). Further data indicates that uninfected but primed or activated T lymphocytes from HIV-infected individuals undergo apoptosis after encountering the TNF-family ligand FasL. Using monocytic cell lines that result in death following HIV infection, it has been demonstrated that infection of U937 cells with HIV results in the de novo expression of FasL and that FasL mediates HIV-induced apoptosis (Badley, A. D. et al., J. Virol. 70:199-206 (1996)). Further the TNF-family ligand was detectable in uninfected macrophages and its expression was upregulated following HIV infection resulting in selective killing of uninfected CD4+ T-lymphocytes (Badley, A. D et al., J. Virol. 70:199-206 (1996)).

As shown in Example 6, the TR2 receptor shown in FIG. 1A-1B is expressed in CD4+ T-lymphocytes and is capable of inducing lymphocyte proliferation. Thus, by the invention, a method for treating HIV+ individuals is provided which involves administering an agonist of the present invention to increase the rate of proliferation and differentiation of CD4+ T-lymphocytes. Such agonists include agents capable of inducing the expression of TR2 receptors (e.g., TNFα, PMA and DMSO) or enhancing the signal of such receptors which induces lymphocyte proliferation and differentiation. Modes of administration and dosages are discussed in detail below.

In rejection of an allograft, the immune system of the recipient animal has not previously been primed to respond because the immune system for the most part is only primed by environmental antigens. Tissues from other members of the same species have not been presented in the same way that, for example, viruses and bacteria have been presented. In the case of allograft rejection, immunosuppressive regimens are designed to prevent the immune system from reaching the effector stage. However, the immune profile of xenograft rejection may resemble disease recurrence more that allograft rejection. In the case of disease recurrence, the immune system has already been activated, as evidenced by destruction of the native islet cells. Therefore, in disease recurrence the immune system is already at the effector stage. Antagonists of the present invention are able to suppress the immune response to both allografts and xenografts by decreasing the rate of TR2 mediated lymphocyte proliferation and differentiation. Such antagonists include the TR2-Fc fusion protein described in Examples 5 and 6. Thus, the present invention further provides a method for suppression of immune responses.

In addition, TNF-α has been shown to prevent diabetes in strains of animals which are prone to this affliction resulting from autoimmunity. See Porter, A., Tibtech 9:158-162 (1991). Thus, agonists and antagonists of the present invention may be useful in the treatment of autoimmune diseases such as type 1 diabetes.

In addition, the role played by the TR2 receptors in cell proliferation and differentiation indicates that agonist or antagonist of the present invention may be used to treat disease states involving aberrant cellular expression of these receptors. TR2 receptors may in some circumstances induce an inflammatory response, and antagonists may be useful reagents for blocking this response. Thus TR2 receptor antagonists (e.g., soluble forms of the TR2 receptors; neutralizing antibodies) may be useful for treating inflammatory diseases, such as rheumatoid arthritis, osteoarthritis, psoriasis, septicemia, and inflammatory bowel disease.

Antagonists to the TR2 receptor may also be employed to treat and/or prevent septic shock, which remains a critical clinical condition. Septic shock results from an exaggerated host response, mediated by protein factors such as TNF and IL-1, rather than from a pathogen directly. For example, lipopolysaccharides have been shown to elicit the release of TNF leading to a strong and transient increase of its serum concentration. TNF causes shock and tissue injury when administered in excessive amounts. Accordingly, it is believed that antagonists to the TR2 receptor will block the actions of TNF and treat/prevent septic shock. These antagonists may also be employed to treat meningococcemia in children which correlates with high serum levels of TNF.

Among other disorders which may be treated by the antagonists to TR2 receptors, there are included, inflammation which is mediated by TNF receptor ligands, and the bacterial infections cachexia and cerebral malaria. The TR2 receptor antagonists may also be employed to treat inflammation mediated by ligands to the receptor such as TNF.

In specific embodiments, antagonists according to the present invention are nucleic acids corresponding to the sequences contained in SEQ ID NO:25, FIG. 1A-1B (SEQ ID NO:1), FIG. 4A-4B (SEQ ID NO:4) or FIG. 7A-7B (SEQ ID NO:7) or the complementary strand thereof, and/or to the deposited nucleotide sequences of ATCC Deposit Numbers 97059, 97058 or 97057. In one embodiment, antisense sequence is generated internally by the organism, in another embodiment, the antisense sequence is separately administered (see, e.g, O'Connor, J. Neurochem. 56:560 (1991), and Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988). Antisense technology can be used to control gene expression through antisense DNA or RNA, or through triple-helix formation. Antisense techniques are discussed for example, in Okano, J. Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988). Triple helix formation is discussed in, for instance, Lee et al., Nucleic Acids Research 6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al., Science 251:1300 (1991). The methods are based on binding of a polynucleotide to a complementary DNA or RNA.

For example, the 5′ coding portion of a polynucleotide that encodes the mature polypeptide of the present invention may be used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription thereby preventing transcription and the production of the receptor. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into receptor polypeptide.

In one embodiment, the TR2 receptor antisense nucleic acid of the invention is produced intracellularly by transcription from an exogenous sequence. For example, a vector or a portion thereof, is transcribed, producing an antisense nucleic acid (RNA) of the invention. Such a vector would contain a sequence encoding the TR2 receptor antisense nucleic acid. Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be plasmid, viral, or others know in the art, used for replication and expression in vertebrate cells. Expression of the sequence encoding a TR2 receptor, or fragments thereof, can be by any promoter known in the art to act in vertebrate, preferably human cells. Such promoters can be inducible or constitutive. Such promoters include, but are not limited to, the SV40 early promoter region (Bernoist and Chambon, Nature 29:304-310 (1981), the promoter contained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell 22:787-797 (1980), the Herpes thymidine promoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445 (1981), the regulatory sequences of the metallothionein gene (Brinster, et al., Nature 296:39-42 (1982)), etc.

The antisense nucleic acids of the invention comprise a sequence complementary to at least a portion of an RNA transcript of a TR2 receptor gene. However, absolute complementarity, although preferred, is not required. A sequence “complementary to at least a portion of an RNA,” referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double stranded TR2 receptor antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid Generally, the larger the hybridizing nucleic acid, the more base mismatches with a TR2 receptor RNA it may contain and still form a stable duplex (or triplex as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.

Oligonucleotides that are complementary to the 5′ end of the message, e.g., the 5′ untranslated sequence up to and including the AUG initiation codon, should work most efficiently at inhibiting translation. However, sequences complementary to the 3′ untranslated sequences of mRNAs have been shown to be effective at inhibiting translation of mRNAs as well. See generally, Wagner, R., Nature 372:333-335 (1994). Thus, oligonucleotides complementary to either the 5′- or 3′-non-translated, non-coding regions of the TR2 receptor shown in SEQ ID NO:25, FIG. 1A-1B (SEQ ID NO:1), FIG. 4A-4B (SEQ ID NO:4) or FIG. 7A-7B (SEQ ID NO:7) could be used in an antisense approach to inhibit translation of endogenous TR2 receptor mRNA. Oligonucleotides complementary to the 5′ untranslated region of the mRNA should include the complement of the AUG start codon. Antisense oligonucleotides complementary to mRNA coding regions are less efficient inhibitors of translation but could be used in accordance with the invention. Whether designed to hybridize to the 5′-, 3′- or coding region of TR2 receptor mRNA, antisense nucleic acids should be at least six nucleotides in length, and are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length. In specific aspects the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides.

The polynucleotides of the invention can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc. The oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556 (1989); Lemaitre et al., Proc. Natl. Acad. Sci. 84:648-652 (1987); PCT Publication No. WO88/09810, published Dec. 15, 1988) or the blood-brain barrier (see, e.g., PCT Publication No. WO89/10134, published Apr. 25, 1988), hybridization-triggered cleavage agents. (See, e.g., Krol et al., BioTechniques 6:958-976 (1988)) or intercalating agents. (See, e.g., Zon, Pharm. Res. 5:539-549 (1988)). To this end, the oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.

The antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including, but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.

The antisense oligonucleotide may also comprise at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide comprises at least one modified phosphate backbone selected from the group including, but not limited to, a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.

In yet another embodiment, the antisense oligonucleotide is an α-anomeric oligonucleotide. An α-anomenic oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gautier et al., Nucl. Acids Res. 15:6625-6641 (1987)). The oligonucleotide is a 2′-0-methylribonucleotide (Inoue et al., Nucl. Acids Res. 15:6131-6148 (1987)), or a chimeric RNA-DNA analogue (Inoue et al., FEBS Lett. 215:327-330 (1987)).

Polynucleotides of the invention may be synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides may be synthesized by the method of Stein et al. (Nucl. Acids Res. 16:3209 (1988)), methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al., Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451 (1988)), etc.

While antisense nucleotides complementary to TR2 receptor coding region sequences could be used, those complementary to the transcribed untranslated region are most preferred.

Potential antagonists according to the invention also include catalytic RNA, or a ribozyme (See, e.g., PCT International Publication WO 90/11364, published Oct. 4, 1990; Sarver et al., Science 247:1222-1225 (1990). While ribozymes that cleave mRNA at site specific recognition sequences can be used to destroy TR2 receptor mRNAs, the use of hammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA have the following sequence of two bases: 5′-UG-3′. The construction and production of hammerhead ribozymes is well known in the art and is described more fully in Haseloff and Gerlach, Nature 334:585-591 (1988). There are numerous potential hammerhead ribozyme cleavage sites within the nucleotide sequence of the TR2 receptors (SEQ ID NO:25, FIG. 1A-1B (SEQ ID NO:1), FIG. 4A-4B (SEQ ID NO:4) and FIG. 7A-7B (SEQ ID NO:7)). Preferably, the ribozyme is engineered so that the cleavage recognition site is located near the 5′ end of the subject TR2 receptor mRNA; i.e., to increase efficiency and minimize the intracellular accumulation of non-functional mRNA transcripts.

As in the antisense approach, the ribozymes of the invention can be composed of modified oligonucleotides (e.g. for improved stability, targeting, etc.) and should be delivered to cells which express TR2 receptors in vivo. DNA constructs encoding the ribozyme may be introduced into the cell in the same manner as described above for the introduction of antisense encoding DNA. A preferred method of delivery involves using a DNA construct “encoding” the ribozyme under the control of a strong constitutive promoter, such as, for example, pol III or pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous TR2 receptor messages and inhibit translation. Since ribozymes unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency.

The compounds or pharmaceutical compositions of the invention are preferably tested in vitro, and then in vivo for the desired therapeutic or prophylactic activity, prior to use in humans. For example, in vitro assays to demonstrate the therapeutic or prophylactic utility of a compound or pharmaceutical composition include, the effect of a compound on a cell line or a patient tissue sample. The effect of the compound or composition on the cell line and/or tissue sample can be determined utilizing techniques known to those of skill in the art including, but not limited to, inhibition or stimulation of proliferation. In accordance with the invention, in vitro assays which can be used to determine whether administration of a specific compound is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise administered a compound, and the effect of such compound upon the tissue sample is observed.

Endogenous gene expression can also be reduced by inactivating or “knocking out” the TR2 receptor gene and/or its promoter using targeted homologous recombination. (See, e.g., Smithies et al., Nature 317:230-234 (1985); Thomas & Capecchi, Cell 51:503-512 (1987); Thompson et al., Cell 5:313-321 (1989); each of which is incorporated by reference herein in its entirety). For example, a mutant, non-functional polynucleotide of the invention (or a completely unrelated DNA sequence) flanked by DNA homologous to the endogenous polynucleotide sequence (either the coding regions or regulatory regions of the gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express polypeptides of the invention in vivo. In another embodiment, techniques known in the art are used to generate knockouts in cells that contain, but do not express the gene of interest. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation of the targeted gene. Such approaches are particularly suited in research and agricultural fields where modifications to embryonic stem cells can be used to generate animal offspring with an inactive targeted gene (see, e.g., Thomas & Capecchi 1987 and Thompson 1989, supra). However this approach can be routinely adapted for use in humans provided the recombinant DNA constructs are directly administered or targeted to the required site in vivo using appropriate viral vectors that will be apparent to those of skill in the art. The contents of each of the documents recited in this paragraph is herein incorporated by reference in its entirety.

In other embodiments, antagonists according to the present invention include soluble forms of TR2 receptor (e.g., fragments of the TR2 receptors shown in SEQ ID NO:26, FIG. 1A-1B (SEQ ID NO:2), FIG. 4A-4B (SEQ ID NO:5) or FIG. 7A-7B (SEQ ID NO:8)) that include the ligand binding domain from the extracellular region of the full length receptor). Such soluble forms of the TR2 receptor, which may be naturally occurring or synthetic, antagonize TR2 receptor mediated signaling by competing with the cell surface bound forms of the receptor for binding to TNF-family ligands. Antagonists of the present invention also include antibodies specific for TNF-family ligands and TR2 receptor-Fc fusion proteins.

By a “TNF-family ligand” is intended naturally occurring, recombinant, and synthetic ligands that are capable of binding to a member of the TNF receptor family and inducing and/or blocking the ligand/receptor signaling pathway. Members of the TNF ligand family include, but are not limited to, TNF-α, lymphotoxin-α (LT-α, also known as TNF-β), LT-β (found in complex heterotrimer LT-α 2-β), FasL, VEGI (International Publication No. WO 96/14328), AIM I (International Publication No. WO 97/33899), AIM II (International Publication No. WO 97/34911), APRIL (J. Exp. Med. 188(6):1185-1190), endokine-α (International Publication No. WO 98/07880), neutrokine-α (International Publication No. WO 98/18921), CD40L, CD27L, CD30L, 4-1BBL, OX40L and nerve growth factor (NGF).

TNF-α has been shown to protect mice from infection with Herpes simplex virus type 1 (HSV-1). Rossol-Voth et al., J. Gen. Virol. 72:143-147 (1991). The mechanism of the protective effect of TNF-α is unknown but appears to involve neither interferons nor NK cell killing. One member of the family has been shown to mediate HSV-1 entry into cells. Montgomery et al., Eur. Cytokine Newt. 7:159 (1996). Further, antibodies specific for the extracellular domain of this block HSV-1 entry into cells. Thus, TR2 receptor antagonists of the present invention include both TR2 receptor amino acid sequences and antibodies capable of preventing mediated viral entry into cells. Such sequences and antibodies can function by either competing with cell surface localized for binding to virus or by directly blocking binding of virus to cell surface receptors.

Antibodies according to the present invention may be prepared by any of a variety of standard methods using TR2 receptor immunogens of the present invention. Such TR2 receptor immunogens include the TR2 receptor proteins shown in SEQ ID NO:26, FIG. 1A-1B (SEQ ID NO:2), FIG. 4A-4B (SEQ ID NO:5) and FIG. 7A-7B (SEQ ID NO:8) (which may or may not include a leader sequence) and polypeptide fragments of the receptor comprising the ligand binding, extracellular (e.g., one or more of the cysteine repeat regions), transmembrane, the intracellular domains of TR2 receptor, or any combination thereof.

Polyclonal and monoclonal antibody agonists or antagonists according to the present invention can be raised according to the methods disclosed herein and/or known in the art, such as, for example, those methods described in Tartaglia and Goeddel, J. Biol. Chem. 267:4304-4307 (1992)); Tartaglia et al., Cell 73:213-216 (1993)), and PCT Application WO 94/09137 (the contents of each of these three applications are herein incorporated by reference in their entireties), and are preferably specific to polypeptides of the invention having the amino acid sequence of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, or SEQ ID NO:26.

The techniques of gene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling (collectively referred to as “DNA shuffling”) may be employed to modulate the activities of TR2 thereby effectively generating agonists and antagonists of TR2. See generally, U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458, and Patten, P. A., et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, S., Trends Biotechnol. 16(2):76-82 (1998); Hansson, L. O. et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo, M. M. and Blasco, R., BioTechniques 24(2): 308-13 (1998) (each of these patents and publications are hereby incorporated by reference). In one embodiment, alteration of TR2 polynucleotides and corresponding polypeptides may be achieved by DNA shuffling. DNA shuffling involves the assembly of two or more DNA segments into a desired TR2 molecule by homologous, or site-specific, recombination. In another embodiment, TR2 polynucleotides and corresponding polypeptides may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination. In another embodiment, one or more components, motifs, sections, parts, domains, fragments, etc., of TR2 may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules. In preferred embodiments, the heterologous molecules are, for example, TNF-alpha, lymphotoxin-αlpha (LT-alpha, also known as TNF-beta), LT-beta (found in complex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF-gamma (International Publication No. WO 96/14328), AIM I (International Publication No. WO 97/33899), AIM II (International Publication No. WO 97/34911), APRIL (J. Exp. Med. 188(6):1185-1190), endokine-alpha (International Publication No. WO 98/07880), Neutrokine-alpha (International Publication No. WO 98/18921), OPG, and neutrokine-alpha (International Publication No. WO 98/18921, OX40, and nerve growth factor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, DR3 (International Publication No. WO 97/33904), DR4 (International Publication No. WO 98/32856), TR5 (International Publication No. WO 98/30693), TR6 (International Publication No. WO 98/30694) TR7 (International Publication No. WO 98/41629), TRANK, TR9 (International Publication No. WO 98/56892), TR10 (International Publication No. WO 98/54202), 312C2 (International Publication No. WO 98/06842), TR12, and TNF-R1, TRAMP/DR3/APO-3/WSL/LARD, TRAIL-R1/DR4/APO-2, TRAIL-R2/DR5, DcR1/TRAIL-R3/TRID/LIT, DcR2/TRAIL-R4, CAD, TRAIL, TRAMP, v-FLIP.

In further preferred embodiments, the heterologous molecules are any member of the TNF family.

Therapeutic and Other Uses

The Tumor Necrosis Factor (TNF) family ligands are known to be among the most pleiotropic cytokines, inducing a large number of cellular responses, including cytotoxicity, anti-viral activity, immunoregulatory activities, and the transcriptional regulation of several genes (Goeddel, D. V., et al., “Tumor Necrosis Factors Gene Structure and Biological Activities,” Symp. Quant. Biol. 51:597-609, Cold Spring Harbor (1986); Beutler, B., and Cerami, A., Annu. Rev. Biochem. 57:505-518 (1988); Old, L. J., Sci. Am. 258:59-75 (1988); Fiers, W., FEBS Lett. 285:199-224 (1991)). The TNF-family ligands induce such various cellular responses by binding to TNF-family receptors.

TR2 polynucleotides or polypeptides, or agonists of TR2, can be used in the treatment of infectious agents. For example, by increasing the immune response, particularly increasing the proliferation and differentiation of B cells, infectious diseases may be treated. The immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response. Alternatively, TR2 polynucleotides or polypeptides, or agonists or antagonists of TR2, may also directly inhibit the infectious agent, without necessarily eliciting an immune response.

As noted above, TR2 polynucleotides and polypeptides, and anti-TR2 antibodies, are useful for diagnosis of conditions involving abnormally high or low expression of TR2, TR2-SV1 and/or TR2-SV2 and/or TR2, TR2-SV1 and/or TR2-SV2 activities. Given the cells and tissues where TR2, TR2-SV1 and/or TR2-SV2 is expressed as well as the activities modulated by TR2, TR2-SV1 and/or TR2-SV2, it is readily apparent that a substantially altered (increased or decreased) level of expression of TR2, TR2-SV1 and/or TR2-SV2 in an individual compared to the standard or “normal” level produces pathological conditions related to the bodily system(s) in which TR2, TR2-SV1 and/or TR2-SV2 is expressed and/or is active.

It will also be appreciated by one of ordinary skill that, since the TR2 polypeptides of the invention are members of the TNF family, the extracellular domains of the respective proteins may be released in soluble form from the cells which express TR2, TR2-SV1 and/or TR2-SV2 by proteolytic cleavage and therefore, when TR2, TR2-SV1 and/or TR2-SV2 polypeptide (particularly a soluble form of the respective extracellular domains) is added from an exogenous source to cells, tissues or the body of an individual, the polypeptide will exert its modulating activities on any of its target cells of that individual. Also, cells expressing this type II transmembrane protein may be added to cells, tissues or the body of an individual whereby the added cells will bind to cells expressing receptor for TR2, TR2-SV1 and/or TR2-SV2 whereby the cells expressing TR2, TR2-SV1 and/or TR2-SV2 can cause actions (e.g., reduced proliferation or cytotoxicity) on the receptor-bearing target cells.

In one embodiment, the invention provides a method of delivering compositions containing the polypeptides of the invention (e.g., compositions containing TR2, TR2-SV1 and/or TR2-SV2 polypeptides or anti-TR2, anti-TR2-SV1 and/or anti-TR2-SV2 antibodies associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs) to targeted cells, such as, for example, B cells expressing a TR2, TR2-SV1 and/or TR2-SV2 receptor, or monocytes expressing the cell surface bound form of TR2, TR2-SV1 and/or TR2-SV2. TR2, TR2-SV1 and/or TR2-SV2 polypeptides or anti-TR2, anti-TR2-SV1 and/or anti-TR2-SV2 antibodies of the invention may be associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalent interactions.

In one embodiment, the invention provides a method for the specific delivery of compositions of the invention to cells by administering polypeptides of the invention (e.g., TR2, TR2-SV1 and/or TR2-SV2 polypeptides or anti-TR2, anti-TR2-SV1 and/or anti-TR2-SV2 antibodies) that are associated with heterologous polypeptides or nucleic acids. In one example, the invention provides a method for delivering a therapeutic protein into the targeted cell. In another example, the invention provides a method for delivering a single stranded nucleic acid (e.g., antisense or ribozymes) or double stranded nucleic acid (e.g., DNA that can integrate into the cell's genome or replicate episomally and that can be transcribed) into the targeted cell.

In another embodiment, the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides of the invention (e.g., TR2, TR2-SV1 and/or TR2-SV2 polypeptides or anti-TR2, anti-TR2-SV1 and/or anti-TR2-SV2 antibodies) in association with toxins or cytotoxic prodrugs.

In a specific embodiment, the invention provides a method for the specific destruction of cells of B cell lineage (e.g., B cell related leukemias or lymphomas) by administering TR2, TR2-SV1 and/or TR2-SV2 polypeptides and/or anti-TR2 antibodies in association with toxins or cytotoxic prodrugs.

By “toxin” is meant compounds that bind and activate endogenous cytotoxic effector systems, radioisotopes, holotoxins, modified toxins, catalytic subunits of toxins, cytotoxins (cytotoxic agents), or any molecules or enzymes not normally present in or on the surface of a cell that under defined conditions cause the cell's death. Toxins that may be used according to the methods of the invention include, but are not limited to, radioisotopes known in the art, compounds such as, for example, antibodies (or complement fixing containing portions thereof) that bind an inherent or induced endogenous cytotoxic effector system, thymidine kinase, endonuclease, RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral protein, alpha-sarcin and cholera toxin. “Toxin” also includes a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters such as, for example, 213Bi, or other radioisotopes such as, for example, 103Pd, 133Xe, 131I, 68Ge, 57Co, 65Zn, 85Sr, 32P, 35S, 90Y, 153Sm, 153Gd, 169Yb, 51Cr, 54Mn, Se, 113Sn, 90Yttrium, 117Tin, 186Rhenium, 166Holmium, and 188Rhenium; luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.

Techniques known in the art may be applied to label antibodies of the invention. Such techniques include, but are not limited to, the use of bifunctional conjugating agents (see e.g., U.S. Pat. Nos. 5,756,065; 5,714,631; 5,696,239; 5,652,361; 5,505,931; 5,489,425; 5,435,990; 5,428,139; 5,342,604; 5,274,119; 4,994,560; and 5,808,003; the contents of each of which are hereby incorporated by reference in its entirety). A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, cis-dichlorodiamine platinum (II) (DDP), cisplatin, anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).

By “cytotoxic prodrug” is meant a non-toxic compound that is converted by an enzyme, normally present in the cell, into a cytotoxic compound. Cytotoxic prodrugs that may be used according to the methods of the invention include, but are not limited to, glutamyl derivatives of benzoic acid mustard alkylating agent, phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside, daunorubisin, and phenoxyacetamide derivatives of doxorubicin.

It will be appreciated that conditions caused by a decrease in the standard or normal level of TR2, TR2-SV1 and/or TR2-SV2 activity in an individual, particularly disorders of the immune system, can be treated by administration of TR2, TR2-SV1 and/or TR2-SV2 polypeptide (in the form of soluble extracellular domain or cells expressing the complete protein) or agonist. Thus, the invention also provides a method of treatment of an individual in need of an increased level of TR2, TR2-SV1 and/or TR2-SV2 activity comprising administering to such an individual a pharmaceutical composition comprising an amount of an isolated TR2, TR2-SV1 and/or TR2-SV2 polypeptide of the invention, or agonist thereof, effective to increase the TR2, TR2-SV1 and/or TR2-SV2 activity level in such an individual.

It will also be appreciated that conditions caused by a increase in the standard or normal level of TR2, TR2-SV1 and/or TR2-SV2 activity in an individual, particularly disorders of the immune system, can be treated by administration of TR2, TR2-SV1 and/or TR2-SV2 polypeptides (in the form of soluble extracellular domain or cells expressing the complete protein) or antagonist (e.g., an anti-TR2 antibody). Thus, the invention also provides a method of treatment of an individual in need of an decreased level of TR2, TR2-SV1 and/or TR2-SV2 activity comprising administering to such an individual a pharmaceutical composition comprising an amount of an isolated TR2, TR2-SV1 and/or TR2-SV2 polypeptide of the invention, or antagonist thereof, effective to decrease the TR2, TR2-SV1 and/or TR2-SV2 activity level in such an individual.

TR2 polynucleotides or polypeptides of the invention, or agonists or antagonists of TR2 can be used in the treatment of infectious agents. For example, by increasing the immune response, particularly increasing the proliferation and differentiation of B cells, infectious diseases may be treated. The immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response. Alternatively, TR2 polynucleotides or polypeptides, or agonists of TR2 may also directly inhibit the infectious agent, without necessarily eliciting an immune response.

Viruses are one example of an infectious agent that can cause disease or symptoms that can be treated by TR2, TR2-SV1 and/or TR2-SV2 polynucleotides or polypeptides, or agonists of TR2, TR2-SV1 and/or TR2-SV2. Examples of viruses, include, but are not limited to the following DNA and RNA viruses and viral families: Arbovirus, Adenoviridae, Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Dengue, EBV, HIV, Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae (such as, Cytomegalovirus, Herpes Simplex, Herpes Zoster), Mononegavirus (e.g., Paramyxoviridae, Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g., Influenza A, Influenza B, and parainfluenza), Papiloma virus, Papovaviridae, Parvoviridae, Picornaviridae, Poxyiridae (such as Smallpox or Vaccinia), Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentivirus), and Togaviridae (e.g., Rubivirus). Viruses falling within these families can cause a variety of diseases or symptoms, including, but not limited to: arthritis, bronchiollitis, respiratory syncytial virus, encephalitis, eye infections (e.g., conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active, Delta), Japanese B encephalitis, Junin, Chikungunya, Rift Valley fever, yellow fever, meningitis, opportunistic infections (e.g., AIDS), pneumonia, Burkitt's Lymphoma, chickenpox, hemorrhagic fever, Measles, Mumps, Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella, sexually transmitted diseases, skin diseases (e.g., Kaposi's, warts), and viremia. TR2, TR2-SV1 and/or TR2-SV2 polynucleotides or polypeptides, or agonists or antagonists of TR2, TR2-SV1 and/or TR2-SV2, can be used to treat, prevent, diagnose, and/or detect any of these symptoms or diseases. In specific embodiments, TR2, TR2-SV1 and/or TR2-SV2 polynucleotides, polypeptides, or agonists are used to treat, prevent, and/or diagnose: meningitis, Dengue, EBV, and/or hepatitis (e.g., hepatitis B). In an additional specific embodiment TR2, TR2-SV1 and/or TR2-SV2 polynucleotides, polypeptides, or agonists are used to treat patients nonresponsive to one or more other commercially available hepatitis vaccines. In a further specific embodiment, TR2, TR2-SV1 and/or TR2-SV2 polynucleotides, polypeptides, or agonists are used to treat, prevent, and/or diagnose AIDS. In an additional specific embodiment TR2, TR2-SV1 and/or TR2-SV2 receptor polynucleotides, polypeptides, agonists, and/or antagonists are used to treat, prevent, and/or diagnose patients with cryptosporidiosis.

Similarly, bacterial or fungal agents that can cause disease or symptoms and that can be treated by TR2, TR2-SV1 and/or TR2-SV2 polynucleotides or polypeptides, or agonists or antagonists of TR2, TR2-SV1 and/or TR2-SV2, include, but not limited to, the following Gram-Negative and Gram-positive bacteria and bacterial families and fungi: Actinomycetales (e.g., Corynebacterium, Mycobacterium, Norcardia), Cryptococcus neoformans, Aspergillosis, Bacillaceae (e.g., Anthrax, Clostridium), Bacteroidaceae, Blastomycosis, Bordetella, Borrelia (e.g., Borrelia burgdorferi, Brucellosis, Candidiasis, Campylobacter, Coccidioidomycosis, Cryptococcosis, Dermatocycoses, E. coli (e.g., Enterotoxigenic E. coli and Enterohemorrhagic E. coli), Enterobacteriaceae (Klebsiella, Salmonella (e.g., Salmonella typhi and Salmonella paratyphi), Serratia, Yersinia), Erysipelothrix, Helicobacter, Legionellosis, Leptospirosis, Listeria (e.g., Listeria monocytogenes), Mycoplasmatales, Mycobacterium leprae, Vibrio cholerae, Neisseriaceae (e.g., Acinetobacter, Gonorrhea, Menigococcal), Meisseria meningitidis, Pasteurellacea Infections (e.g., Actinobacillus, Heamophilus (e.g., Heamophilusinfluenza type B), Pasteurella), Pseudomonas, Rickettsiaceae, Chlamydiaceae, Syphilis, Shigella spp., Staphylococcal, Meningiococcal, Pneumococcal and Streptococcal (e.g., Streptococcus pneumoniae and Group B Streptococcus). These bacterial or fungal families can cause the following diseases or symptoms, including, but not limited to: bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis, uveitis), gingivitis, opportunistic infections (e.g., AIDS related infections), paronychia, prosthesis-related infections, Reiter's Disease, respiratory tract infections, such as Whooping Cough or Empyema, sepsis, Lyme Disease, Cat-Scratch Disease, Dysentery, Paratyphoid Fever, food poisoning, Typhoid, pneumonia, Gonorrhea, meningitis (e.g., mengitis types A and B), Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis, Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, Rheumatic Fever, Scarlet Fever, sexually transmitted diseases, skin diseases (e.g., cellulitis, dermatocycoses), toxemia, urinary tract infections, wound infections. TR2, TR2-SV1 and/or TR2-SV2 polynucleotides or polypeptides, or agonists or antagonists of TR2, TR2-SV1 and/or TR2-SV2, can be used to treat, prevent, diagnose, and/or detect any of these symptoms or diseases. In specific embodiments, TR2 polynucleotides, polypeptides, or agonists thereof are used to treat, prevent, and/or diagnose: tetanus, Diphtheria, botulism, and/or meningitis type B.

Moreover, parasitic agents causing disease or symptoms that can be treated by TR2, TR2-SV1 and/or TR2-SV2 polynucleotides or polypeptides, or agonists of TR2, TR2-SV1 and/or TR2-SV2, include, but not limited to, the following families or class: Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic, Giardiasis, Helminthiasis, Leishmaniasis, Theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomonas and Sporozoans (e.g., Plasmodium virax, Plasmodium falciparium, Plasmodium malariae and Plasmodium ovale). These parasites can cause a variety of diseases or symptoms, including, but not limited to: Scabies, Trombiculiasis, eye infections, intestinal disease (e.g., dysentery, giardiasis), liver disease, lung disease, opportunistic infections (e.g., AIDS related), malaria, pregnancy complications, and toxoplasmosis. TR2, TR2-SV1 and/or TR2-SV2 polynucleotides or polypeptides, or agonists or antagonists of TR2, TR2-SV1 and/or TR2-SV2, can be used to treat, prevent, diagnose, and/or detect any of these symptoms or diseases. In specific embodiments, TR2, TR2-SV1 and/or TR2-SV2 polynucleotides, polypeptides, or agonists thereof are used to treat, prevent, and/or diagnose malaria.

TR2 receptor polynucleotides, polypeptides, agonists or antagonists of the invention may be used in developing treatments for any disorder mediated (directly or indirectly) by defective, or insufficient amounts of TR2. TR2, TR2-SV1 and/or TR2-SV2 receptor polypeptides, agonists or antagonists may be administered to a patient (e.g., mammal, preferably human) afflicted with such a disorder. Alternatively, a gene therapy approach may be applied to treat such disorders. Disclosure herein of TR2 receptor nucleotide sequences permits the detection of defective TR2 receptor genes, and the replacement thereof with normal TR2 receptor-encoding genes. Defective genes may be detected in in vitro diagnostic assays, and by comparison of the TR2 receptor nucleotide sequence disclosed herein with that of a TR2 receptor gene derived from a patient suspected of harboring a defect in this gene.

In another embodiment, the polypeptides of the present invention are used as a research tool for studying the biological effects that result from inhibiting AIM II/TR2 receptor and/or lymphotoxin-α/TR2 receptor interactions on different cell types. TR2 receptor polypeptides also may be employed in in vitro assays for detecting AIM II, lymphotoxin-α or TR2 receptor or the interactions thereof.

In another embodiment, a purified TR2 receptor polypeptide or antagonist is used to inhibit binding of AIM-II or lymphotoxin-α to endogenous cell surface AIM II and/or lymphotoxin-α receptors. Certain ligands of the TNF family (of which AIM II and lymphotoxin-α are members) have been reported to bind to more than one distinct cell surface receptor protein. AIM II and lymphotoxin-α likewise are believed to bind multiple cell surface proteins. By binding AIM II and/or lymphotoxin-α, soluble TR2 receptor polypeptides of the present invention may be employed to inhibit the binding of AIM II and/or lymphotoxin-α not only to cell surface TR2 receptor, but also to AIM II and/or lymphotoxin-α receptor proteins that are distinct from TR2 receptor. Thus, in another embodiment, TR2 receptor polynucleotides, polypeptides, agonists or antagonists are used to inhibit a biological activity of AIM II and/or lymphotoxin-α, in in vitro or in vivo procedures. By inhibiting binding of AIM II and/or lymphotoxin-α to cell surface receptors, TR2 receptor polynucleotides, polypeptides, agonists or antagonists also inhibit biological effects that result from the binding of AIM II and/or lymphotoxin-α to endogenous receptors. Various forms of TR2 receptor may be employed, including, for example, the above-described TR2 receptor fragments, derivatives, and variants that are capable of binding AIM II and/or lymphotoxin-α. In one preferred embodiment, a soluble TR2 receptor polypeptide is employed to inhibit a biological activity of AIM-II (e.g., to inhibit AIM II-mediated apoptosis of cells susceptible to such apoptosis). In another preferred embodiment, a soluble TR2 receptor polypeptide is employed to inhibit a biological activity of lymphotoxin-α (e.g., induction of inflammation and immune responses, maintenance of lymphoid tissues, induction of B cell proliferation).

In a further embodiment, a TR2 receptor polynucleotide, polypeptide, agonist or antagonist is administered to a mammal (e.g., a human) to treat a AIM II-mediated and/or lymphotoxin-α mediated disorder. Such AIM II-mediated and/or lymphotoxin-α mediated disorders include conditions caused (directly or indirectly) or exacerbated by AIM II and/or lymphotoxin-α.

Diseases associated with increased cell survival, or the inhibition of apoptosis, include cancers (such as follicular lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors, including, but not limited to colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Karposi's sarcoma and ovarian cancer); autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome, Grave's disease, Hashimoto's thyroiditis, autoimmune diabetes, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis, autoimmune gastritis, autoimmune thrombocytopenic purpura, and rheumatoid arthritis) and viral infections (such as herpes viruses, pox viruses and adenoviruses), inflammation, graft vs. host disease (acute and/or chronic), acute graft rejection, and chronic graft rejection. In preferred embodiments, TR2 receptor polynucleotides, polypeptides, agonists, or antagonists of the invention are used to inhibit growth, progression, and/or metastasis of cancers, in particular those listed above or in the paragraph that follows.

Additional diseases or conditions associated with increased cell survival include, but are not limited to, progression, and/or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.

Diseases associated with increased apoptosis include AIDS; neurodegenerative disorders (such as Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration and brain tumor or prior associated disease); autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome, Grave's disease Hashimoto's thyroiditis, autoimmune diabetes, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus, immune-related glomerulonephritis, autoimmune gastritis, thrombocytopenic purpura, and rheumatoid arthritis) myelodysplastic syndromes (such as aplastic anemia), graft vs. host disease (acute and/or chronic), ischemic injury (such as that caused by myocardial infarction, stroke and reperfusion injury), liver injury or disease (e.g., hepatitis related liver injury, cirrhosis, ischemia/reperfusion injury, cholestosis (bile duct injury) and liver cancer); toxin-induced liver disease (such as that caused by alcohol), septic shock, ulcerative colitis, cachexia and anorexia. In preferred embodiments, TR2 receptor polynucleotides, polypeptides, agonists, and/or antagonists are used to treat the diseases and disorders listed above.

Many of the pathologies associated with HIV are mediated by apoptosis, including HIV-induced nephropathy and HIV encephalitis. Thus, in additional preferred embodiments, TR2 receptor polynucleotides, polypeptides, agonists or antagonists of the invention are used to treat AIDS and pathologies associated with AIDS.

Another embodiment of the present invention is directed to the use of TR2 receptor polynucleotides, polypeptides, agonists or antagonists to reduce AIM II-mediated death of T cells in HIV-infected patients. The role of T cell apoptosis in the development of AIDS has been the subject of a number of studies (see, for example, Meyaard et al., Science 257:217-219 (1992); Groux et al., J. Exp. Med. 175:331 (1992); and Oyaizu et al., in “Cell Activation and Apoptosis in HIV Infection,” Andrieu and Lu, eds., Plenum Press, New York, pp. 101-114 (1995). Fas-mediated apoptosis has been implicated in the loss of T cells in HIV individuals (Katsikis et al., J. Exp. Med. 181:2029-2036 (1995). It is also likely that T cell apoptosis occurs through multiple mechanisms. For example, at least some of the T cell death seen in HIV patients is likely to be mediated by AIM II.

Activated human T cells are induced to undergo programmed cell death (apoptosis) upon triggering through the CD3/T cell receptor complex, a process termed activated-induced cell death (AICD). AICD of CD4 T cells isolated from HIV-Infected asymptomatic individuals has been reported (Groux et al., supra). Thus, AICD may play a role in the depletion of CD4+ T cells and the progression to AIDS in HIV-infected individuals. Thus, the present invention provides a method of inhibiting AIM II-mediated T cell death in HIV patients, comprising administering a TR2 receptor polynucleotides, polypeptides, agonists or antagonists of the invention (preferably, a soluble TR2 receptor polypeptide) to the patients. In one embodiment, the patient is asymptomatic when treatment with TR2 receptor polynucleotides, polypeptides, agonists or antagonists commences. If desired, prior to treatment, peripheral blood T cells may be extracted from an HIV patient, and tested for susceptibility to AIM II-mediated cell death by procedures known in the art. In one embodiment, a patient's blood or plasma is contacted with TR2 receptor polypeptides of the invention ex vivo. The TR2 receptor polypeptides may be bound to a suitable chromatography matrix by procedures known in the art. The patient's blood or plasma flows through a chromatography column containing TR2 receptor polypeptide bound to the matrix, before being returned to the patient. The immobilized TR2 receptor polypeptide binds AIM II, thus removing AIM-II protein from the patient's blood.

In additional embodiments a TR2 receptor polynucleotide, polypeptide, agonist or antagonist of the invention is administered in combination with other inhibitors of T cell apoptosis. For example, as discussed above, Fas-mediated apoptosis also has been implicated in loss of T cells in HIV individuals (Katsikis et al., J. Exp. Med. 181:2029-2036 (1995). Thus, a patient susceptible to both Fas ligand mediated and AIM II mediated T cell death may be treated with both an agent that blocks AIM II/AIM II receptor interactions and an agent that blocks Fas-ligand/Fas interactions. Suitable agents for blocking binding of Fas-ligand to Fas include, but are not limited to, soluble Fas polypeptides; multimeric forms of soluble Fas polypeptides (e.g., dimers of sFas/Fc); anti-Fas antibodies that bind Fas without transducing the biological signal that results in apoptosis; anti-Fas-ligand antibodies that block binding of Fas-ligand to Fas; and muteins of Fas-ligand that bind Fas but do not transduce the biological signal that results in apoptosis. Preferably, the antibodies employed according to this method are monoclonal antibodies. Examples of suitable agents for blocking Fas-ligand/Fas interactions, including blocking anti-Fas monoclonal antibodies, are described in WO 95/10540, hereby incorporated by reference.

In another example, agents which block binding of TRAIL to a TRAIL receptor are administered with the TR2 receptor polynucleotides, polypeptides, agonists or antagonists of the invention. Such agents include, but are not limited to, soluble TRAIL receptor polypeptides (e.g., a soluble form of OPG, DR4 (WO 98/32856); TR5 (WO 98/30693); DR5 (WO 98/41629); and TR10 (WO 98/54202)); multimeric forms of soluble TRAIL receptor polypeptides; and TRAIL receptor antibodies that bind the TRAIL receptor without transducing the biological signal that results in apoptosis, anti-TRAIL antibodies that block binding of TRAIL to one or more TRAIL receptors, and muteins of TRAIL that bind TRAIL receptors but do not transduce the biological signal that results in apoptosis. Preferably, the antibodies employed according to this method are monoclonal antibodies.

Another embodiment of the present invention is directed to the use of TR2 as a regulator of B cell proliferation and differentiation. The assays and experiments described herein clearly provide the scientific rational for the use of TR2 as a regulator of B cell proliferation and differentiation. The possible uses of the soluble or membrane bound TR2, its native ligand and various ligand antagonists are diverse and include treatment of autoimmune disorders and immunodeficiencies resulting from infection, anti-neoplastic therapy and/or inherited disorders. Moreover, many of the pre-neoplastic monoclonal gammopathies and neoplastic B cell diseases such as multiple myeloma may utilize TR2 or its ligand as either inducing or progressing factors.

Accordingly, TR2 or derived, functional agonists (including anti-TR2 antibodies, soluble forms having amino acids sequences contained in the extracellular domain of TR2 (e.g., TR2-Fc) and TR2 ligands), may find application as the following:

As an agent to direct an individuals immune system towards development of a humoral response (i.e., TH2) as opposed to a TH1 cellular response.

As an antigen for the generation of antibodies to inhibit or enhance TR2 mediated responses.

As a means of activating T cells.

As a means of regulating secreted cytokines that are elicited by TR2.

Antagonists of TR2 include binding and/or inhibitory antibodies, antisense nucleic acids, ribozymes, soluble forms of TR2 (e.g., TR2-fc) and TR2 ligand(s). These would be expected to reverse many of the activities of the receptor described above as well as find clinical or practical application as:

Antagonists of TR2 activities can also be used to treat or prevent Herpes viral infections. Such antagonists include full-length and mature TR2 polypeptides of the invention, TR2 fragments (e.g., soluble fragments), and antibodies having specificity for TR2 polypeptides. While not wishing to be limited to a specific mechanism, TR2 antagonist are believed to function in the treatment or prevention Herpes viral infections by blocking Herpes viral entry into cells.

An additional condition, disease or symptom that can be treated by TR2 polynucleotides or polypeptides, or agonists of TR2, is osteomyelitis.

Preferably, treatment using TR2 polynucleotides or polypeptides, or agonists of TR2, could either be by administering an effective amount of TR2 polypeptide to the patient, or by removing cells from the patient, supplying the cells with TR2 polynucleotide, and returning the engineered cells to the patient (ex vivo therapy). Moreover, as further discussed herein, the TR2 polypeptides or polynucleotides can be used as an adjuvant in a vaccine to raise an immune response against infectious disease.

In another embodiment, TR2, TR2-SV1 and/or TR2-SV2 polynucleotides or polypeptides of the invention and/or agonists and/or antagonists thereof, are used to treat, prevent, and/or diagnose inner ear infection (such as, for example, otitis media), as well as other infections characterized by infection with Streptococcus pneumoniae and other pathogenic organisms.

In a specific embodiment, TR2, TR2-SV1 and/or TR2-SV2 polynucleotides or polypeptides, or agonists or antagonists thereof (e.g., anti-TR2, anti-TR2-SV1 and/or anti-TR2-SV2 antibodies) are used to treat or prevent a disorder characterized by deficient serum immunoglobulin production, recurrent infections, and/or immune system dysfunction. Moreover, TR2, TR2-SV1 and/or TR2-SV2 polynucleotides or polypeptides, or agonists or antagonists thereof (e.g., anti-TR2, anti-TR2-SV1 and/or anti-TR2-SV2 antibodies) may be used to treat or prevent infections of the joints, bones, skin, and/or parotid glands, blood-borne infections (e.g., sepsis, meningitis, septic arthritis, and/or osteomyelitis), autoimmune diseases (e.g., those disclosed herein), inflammatory disorders, and malignancies, and/or any disease or disorder or condition associated with these infections, diseases, disorders and/or malignancies) including, but not limited to, CVID, other primary immune deficiencies, HIV disease, CLL, recurrent bronchitis, sinusitis, otitis media, conjunctivitis, pneumonia, hepatitis, meningitis, herpes zoster (e.g., severe herpes zoster), and/or pheumocystis carnii.

TR2, TR2-SV1 and/or TR2-SV2 polynucleotides or polypeptides of the invention, or agonists or antagonists thereof, may be used to diagnose, prognose, treat or prevent one or more of the following diseases or disorders, or conditions associated therewith: primary immuodeficiencies, immune-mediated thrombocytopenia, Kawasaki syndrome, bone marrow transplant (e.g., recent bone marrow transplant in adults or children), chronic B-cell lymphocytic leukemia, HIV infection (e.g., adult or pediatric HIV infection), chronic inflammatory demyelinating polyneuropathy, and post-transfusion purpura.

Additionally, TR2, TR2-SV1 and/or TR2-SV2 polynucleotides or polypeptides of the invention, or agonists or antagonists thereof, may be used to diagnose, prognose, treat or prevent one or more of the following diseases, disorders, or conditions associated therewith, Guillain-Barre syndrome, anemia (e.g., anemia associated with parvovirus B19, patients with stable multiple myeloma who are at high risk for infection (e.g., recurrent infection), autoimmune hemolytic anemia (e.g., warm-type autoimmune hemolytic anemia), thrombocytopenia (e.g., neonatal thrombocytopenia), and immune-mediated neutropenia), transplantation (e.g., cytamegalovirus (CMV)-negative recipients of CMV-positive organs), hypogammaglobulinemia (e.g., hypogammaglobulinemic neonates with risk factor for infection or morbidity), epilepsy (e.g., intractable epilepsy), systemic vasculitic syndromes, myasthenia gravis (e.g., decompensation in myasthenia gravis), dermatomyositis, and polymyositis.

Additional preferred embodiments of the invention include, but are not limited to, the use of TR2, TR2-SV1 and/or TR2-SV2 polypeptides, TR2, TR2-SV1 and/or TR2-SV2 polynucleotides, and functional agonists thereof, in the following applications:

Administration to an animal (e.g., mouse, rat, rabbit, hamster, guinea pig, pigs, micro-pig, chicken, camel, goat, horse, cow, sheep, dog, cat, non-human primate, and human, most preferably human) to boost the immune system to produce increased quantities of one or more antibodies (e.g., IgG, IgA, IgM, and IgE), to induce higher affinity antibody production (e.g., IgG, IgA, IgM, and IgE), and/or to increase an immune response. In a specific nonexclusive embodiment, TR2, TR2-SV1 and/or TR2-SV2 polypeptides of the invention, and/or agonists thereof, are administered to boost the immune system to produce increased quantities of IgG. In another specific nonexclusive embodiment, TR2, TR2-SV1 and/or TR2-SV2 polypeptides of the invention and/or agonists thereof, are administered to boost the immune system to produce increased quantities of IgA. In another specific nonexclusive embodiment, TR2, TR2-SV1 and/or TR2-SV2 polypeptides of the invention and/or agonists thereof, are administered to boost the immune system to produce increased quantities of IgM.

Administration to an animal (including, but not limited to, those listed above, and also including transgenic animals) incapable of producing functional endogenous antibody molecules or having an otherwise compromised endogenous immune system, but which is capable of producing human immunoglobulin molecules by means of a reconstituted or partially reconstituted immune system from another animal (see, e.g., published PCT Application Nos. WO98/24893, WO/9634096, WO/9633735, and WO/9110741).

A vaccine adjuvant that enhances immune responsiveness to specific antigen. In a specific embodiment, the vaccine adjuvant is a TR2, TR2-SV1 and/or TR2-SV2 polypeptide described herein. In another specific embodiment, the vaccine adjuvant is a TR2, TR2-SV1 and/or TR2-SV2 polynucleotide described herein (i.e., the TR2, TR2-SV1 and/or TR2-SV2 polynucleotide is a genetic vaccine adjuvant). As discussed herein, TR2, TR2-SV1 and/or TR2-SV2 polynucleotides may be administered using techniques known in the art, including but not limited to, liposomal delivery, recombinant vector delivery, injection of naked DNA, and gene gun delivery.

An adjuvant to enhance tumor-specific immune responses.

An adjuvant to enhance anti-viral immune responses. Anti-viral immune responses that may be enhanced using the compositions of the invention as an adjuvant, include, but are not limited to, virus and virus associated diseases or symptoms described herein or otherwise known in the art. In specific embodiments, the compositions of the invention are used as an adjuvant to enhance an immune response to a virus, disease, or symptom selected from the group consisting of: AIDS, meningitis, Dengue, EBV, and hepatitis (e.g., hepatitis B). In another specific embodiment, the compositions of the invention are used as an adjuvant to enhance an immune response to a virus, disease, or symptom selected from the group consisting of: HIV/AIDS, Respiratory syncytial virus, Dengue, Rotavirus, Japanese B encephalitis, Influenza A and B, Parainfluenza, Measles, Cytomegalovirus, Rabies, Junin, Chikungunya, Rift Valley fever, Herpes simplex, and yellow fever. In another specific embodiment, the compositions of the invention are used as an adjuvant to enhance an immune response to the HIV gp120 antigen.

An adjuvant to enhance anti-bacterial or anti-fungal immune responses. Anti-bacterial or anti-fungal immune responses that may be enhanced using the compositions of the invention as an adjuvant, include bacteria or fungus and bacteria or fungus associated diseases or symptoms described herein or otherwise known in the art. In specific embodiments, the compositions of the invention are used as an adjuvant to enhance an immune response to a bacteria or fungus, disease, or symptom selected from the group consisting of: tetanus, Diphtheria, botulism, and meningitis type B. In another specific embodiment, the compositions of the invention are used as an adjuvant to enhance an immune response to a bacteria or fungus, disease, or symptom selected from the group consisting of: Vibrio cholerae, Mycobacterium leprae, Salmonella typhi, Salmonellaparatyphi, Meisseria meningitidis, Streptococcus pneumoniae, Group B streptococcus, Shigella spp., Enterotoxigenic Escherichia coli, Enterohemorrhagic E. coli, Borrelia burgdorferi, and Plasmodium (malaria).

An adjuvant to enhance anti-parasitic immune responses. Anti-parasitic immune responses that may be enhanced using the compositions of the invention as an adjuvant, include parasite and parasite associated diseases or symptoms described herein or otherwise known in the art. In specific embodiments, the compositions of the invention are used as an adjuvant to enhance an immune response to a parasite. In another specific embodiment, the compositions of the invention are used as an adjuvant to enhance an immune response to Plasmodium (malaria).

As a stimulator of B cell responsiveness to pathogens.

As an agent that elevates the immune status of an individual prior to their receipt of immunosuppressive therapies.

As an agent to induce higher affinity antibodies.

As an agent to increase serum immunoglobulin concentrations.

As an agent to accelerate recovery of immunocompromised individuals.

As an agent to boost immunoresponsiveness among aged populations.

As an immune system enhancer prior to, during, or after bone marrow transplant and/or other transplants (e.g., allogeneic or xenogeneic organ transplantation). With respect to transplantation, compositions of the invention may be administered prior to, concomitant with, and/or after transplantation. In a specific embodiment, compositions of the invention are administered after transplantation, prior to the beginning of recovery of T-cell populations. In another specific embodiment, compositions of the invention are first administered after transplantation after the beginning of recovery of T cell populations, but prior to full recovery of B cell populations.

As an agent to boost immunoresponsiveness among B cell immunodeficient individuals, such as, for example, an individual who has undergone a partial or complete splenectomy. B cell immunodeficiencies that may be ameliorated or treated by administering the TR2, TR2-SV1 and/or TR2-SV2 polypeptides or polynucleotides of the invention, or agonists thereof, include, but are not limited to, severe combined immunodeficiency (SCID)-X linked, SCID-autosomal, adenosine deaminase deficiency (ADA deficiency), X-linked agammaglobulinemia (XLA), Bruton's disease, congenital agammaglobulinemia, X-linked infantile agammaglobulinemia, acquired agammaglobulinemia, adult onset agammaglobulinemia, late-onset agammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia, transient hypogammaglobulinemia of infancy, unspecified hypogammaglobulinemia, agammaglobulinemia, common variable immunodeficiency (CVID) (acquired), Wiskott-Aldrich Syndrome (WAS), X-linked immunodeficiency with hyper IgM, non X-linked immunodeficiency with hyper IgM, selective IgA deficiency, IgG subclass deficiency (with or without IgA deficiency), antibody deficiency with normal or elevated Igs, immunodeficiency with thymoma, Ig heavy chain deletions, kappa chain deficiency, B cell lymphoproliferative disorder (BLPD), selective IgM immunodeficiency, recessive agammaglobulinemia (Swiss type), reticular dysgenesis, neonatal neutropenia, severe congenital leukopenia, thymic alymphoplasia-aplasia or dysplasia with immunodeficiency, ataxia-telangiectasia, short limbed dwarfism, X-linked lymphoproliferative syndrome (XLP), Nezelof syndrome-combined immunodeficiency with Igs, purine nucleoside phosphorylase deficiency (PNP), MHC Class II deficiency (Bare Lymphocyte Syndrome) and severe combined immunodeficiency.

As an agent to boost immunoresponsiveness among individuals having an acquired loss of B cell function. Conditions resulting in an acquired loss of B cell function that may be ameliorated or treated by administering the TR2, TR2-SV1 and/or TR2-SV2 polypeptides or polynucleotides of the invention, or agonists thereof, include, but are not limited to, HIV Infection, AIDS, bone marrow transplant, and B cell chronic lymphocytic leukemia (CLL).

As an agent to boost immunoresponsiveness among individuals having a temporary immune deficiency. Conditions resulting in a temporary immune deficiency that may be ameliorated or treated by administering the TR2, TR2-SV1 and/or TR2-SV2 polypeptides or polynucleotides of the invention, or agonists thereof, include, but are not limited to, recovery from viral infections (e.g., influenza), conditions associated with malnutrition, recovery from infectious mononucleosis, or conditions associated with stress, recovery from measles, recovery from blood transfusion, recovery from surgery.

As a regulator of antigen presentation by monocytes, dendritic cells, and/or B-cells. In one embodiment, TR2, TR2-SV1 and/or TR2-SV2 polypeptides (in soluble, membrane-bound or transmembrane forms) or polynucleotides enhance antigen presentation or antagonize antigen presentation in vitro or in vivo. Moreover, in related embodiments, this enhancement or antagonization of antigen presentation may be useful in anti-tumor treatment or to modulate the immune system.

As a mediator of mucosal immune responses. The expression of TR2 by monocytes and the responsiveness of B cells to this factor suggests that it may be involved in exchange of signals between B cells and monocytes or their differentiated progeny. This activity is in many ways analogous to the CD40-CD154 signaling between B cells and T cells. TR2 may therefore be an important regulator of T cell independent immune responses to environmental pathogens. In particular, the unconventional B cell populations (CD5+) that are associated with mucosal sites and responsible for much of the innate immunity in humans may respond to TR2 thereby enhancing an individual's protective immune status.

As a means to induce tumor proliferation and thus make it more susceptible to anti-neoplastic agents. For example, multiple myeloma is a slowly dividing disease and is thus refractory to virtually all anti-neoplastic regimens. If these cells were forced to proliferate more rapidly their susceptibility profile would likely change.

As a B cell specific binding protein to which specific activators or inhibitors of cell growth may be attached. The result would be to focus the activity of such activators or inhibitors onto normal, diseased, or neoplastic B cell populations.

As a means of detecting B-lineage cells by virtue of its specificity. This application may require labeling the protein with biotin or other agents (e.g., as described herein) to afford a means of detection.

As a stimulator of B cell production in pathologies such as AIDS, chronic lymphocyte disorder and/or Common Variable Immunodificiency.

As part of a B cell selection device the function of which is to isolate B cells from a heterogenous mixture of cell types. TR2 could be coupled to a solid support to which B cells would then specifically bind. Unbound cells would be washed out and the bound cells subsequently eluted. A nonlimiting use of this selection would be to allow purging of tumor cells from, for example, bone marrow or peripheral blood prior to transplant.

As a therapy for generation and/or regeneration of lymphoid tissues following surgery, trauma or genetic defect.

As a gene-based therapy for genetically inherited disorders resulting in immuno-incompetence such as observed among SCID patients.

As an antigen for the generation of antibodies to inhibit or enhance TR2, TR2-SV1 and/or TR2-SV2 mediated responses.

As a means of activating monocytes/macrophages to defend against parasitic diseases that effect monocytes such as Leshmania.

As pretreatment of bone marrow samples prior to transplant. Such treatment would increase B cell representation and thus accelerate recover.

As a means of regulating secreted cytokines that are elicited by TR2, TR2-SV1 and/or TR2-SV2.

TR2, TR2-SV1 and/or TR2-SV2 polypeptides or polynucleotides of the invention, or agonists may be used to modulate IgE concentrations in vitro or in vivo.

Additionally, TR2, TR2-SV1 and/or TR2-SV2 polypeptides or polynucleotides of the invention, or agonists thereof, may be used to treat, prevent, and/or diagnose IgE-mediated allergic reactions. Such allergic reactions include, but are not limited to, asthma, rhinitis, and eczema.

In a specific embodiment, TR2, TR2-SV1 and/or TR2-SV2 polypeptides or polynucleotides of the invention, or agonists thereof, is administered to treat, prevent, diagnose, and/or ameliorate selective IgA deficiency.

In another specific embodiment, TR2, TR2-SV1 and/or TR2-SV2 polypeptides or polynucleotides of the invention, or agonists thereof, is administered to treat, prevent, diagnose, and/or ameliorate ataxia-telangiectasia.

In another specific embodiment, TR2, TR2-SV1 and/or TR2-SV2 polypeptides or polynucleotides of the invention, or agonists thereof, is administered to treat, prevent, diagnose, and/or ameliorate common variable immunodeficiency.

In another specific embodiment, TR2, TR2-SV1 and/or TR2-SV2 polypeptides or polynucleotides of the invention, or agonists thereof, is administered to treat, prevent, diagnose, and/or ameliorate X-linked agammaglobulinemia.

In another specific embodiment, TR2, TR2-SV1 and/or TR2-SV2 polypeptides or polynucleotides of the invention, or agonists thereof, is administered to treat, prevent, diagnose, and/or ameliorate severe combined immunodeficiency (SCID).

In another specific embodiment, TR2, TR2-SV1 and/or TR2-SV2 polypeptides or polynucleotides of the invention, or agonists thereof, is administered to treat, prevent, diagnose, and/or ameliorate Wiskott-Aldrich syndrome.

In another specific embodiment, TR2, TR2-SV1 and/or TR2-SV2 polypeptides or polynucleotides of the invention, or agonists thereof, is administered to treat, prevent, diagnose, and/or ameliorate X-linked Ig deficiency with hyper IgM.

In another specific embodiment, TR2, TR2-SV1 and/or TR2-SV2 polypeptides or polynucleotides of the invention, or agonists or antagonists (e.g., anti-TR2 antibodies) thereof, is administered to treat, prevent, and/or diagnose chronic myelogenous leukemia, acute myelogenous leukemia, leukemia, hystiocytic leukemia, monocytic leukemia (e.g., acute monocytic leukemia), leukemic reticulosis, Shilling Type monocytic leukemia, and/or other leukemias derived from monocytes and/or monocytic cells and/or tissues.

In another specific embodiment, TR2, TR2-SV1 and/or TR2-SV2 polypeptides or polynucleotides of the invention, or agonists thereof, is administered to treat, prevent, diagnose, and/or ameliorate monocytic leukemoid reaction, as seen, for example, with tuberculosis.

In another specific embodiment, TR2, TR2-SV1 and/or TR2-SV2 polypeptides or polynucleotides of the invention, or agonists thereof, is administered to treat, prevent, diagnose, and/or ameliorate monocytic leukocytosis, monocytic leukopenia, monocytopenia, and/or monocytosis.

In a specific embodiment, TR2, TR2-SV1 and/or TR2-SV2 polynucleotides or polypeptides of the invention, and/or anti-TR2 antibodies and/or agonists or antagonists thereof, are used to treat, prevent, detect, and/or diagnose primary B lymphocyte disorders and/or diseases, and/or conditions associated therewith. In one embodiment, such primary B lymphocyte disorders, diseases, and/or conditions are characterized by a complete or partial loss of humoral immunity. Primary B lymphocyte disorders, diseases, and/or conditions associated therewith that are characterized by a complete or partial loss of humoral immunity and that may be prevented, treated, detected and/or diagnosed with compositions of the invention include, but are not limited to, X-Linked Agammaglobulinemia (XLA), severe combined immunodeficiency disease (SCID), and selective IgA deficiency.

In a preferred embodiment, TR2, TR2-SV1 and/or TR2-SV2 polynucleotides, polypeptides, and/or agonists and/or antagonists thereof are used to treat, prevent, and/or diagnose diseases or disorders affecting or conditions associated with any one or more of the various mucous membranes of the body. Such diseases or disorders include, but are not limited to, for example, mucositis, mucoclasis, mucocolitis, mucocutaneous leishmaniasis (such as, for example, American leishmaniasis, leishmaniasis americana, nasopharyngeal leishmaniasis, and New World leishmaniasis), mucocutaneous lymph node syndrome (for example, Kawasaki disease), mucoenteritis, mucoepidermoid carcinoma, mucoepidermoid tumor, mucoepithelial dysplasia, mucoid adenocarcinoma, mucoid degeneration, myxoid degeneration; myxomatous degeneration; myxomatosis, mucoid medial degeneration (for example, cystic medial necrosis), mucolipidosis (including, for example, mucolipidosis I, mucolipidosis II, mucolipidosis III, and mucolipidosis IV), mucolysis disorders, mucomembranous enteritis, mucoenteritis, mucopolysaccharidosis (such as, for example, type I mucopolysaccharidosis (i.e., Hurler's syndrome), type IS mucopolysaccharidosis (i.e., Scheie's syndrome or type V mucopolysaccharidosis), type II mucopolysaccharidosis (i.e., Hunter's syndrome), type III mucopolysaccharidosis (i.e., Sanfilippo's syndrome), type IV mucopolysaccharidosis (i.e., Morquio's syndrome), type VI mucopolysaccharidosis (i.e., Maroteaux-Lamy syndrome), type VII mucopolysaccharidosis (i.e, mucopolysaccharidosis due to beta-glucuronidase deficiency), and mucosulfatidosis), mucopolysacchariduria, mucopurulent conjunctivitis, mucopus, mucormycosis (i.e., zygomycosis), mucosal disease (i.e., bovine virus diarrhea), mucous colitis (such as, for example, mucocolitis and myxomembranous colitis), and mucoviscidosis (such as, for example, cystic fibrosis, cystic fibrosis of the pancreas, Clarke-Hadfield syndrome, fibrocystic disease of the pancreas, mucoviscidosis, and viscidosis). In a highly preferred embodiment, TR2, TR2-SV1 and/or TR2-SV2 polynucleotides, polypeptides, and/or agonists and/or antagonists thereof are used to treat, prevent, and/or diagnose mucositis, especially as associated with chemotherapy.

In a preferred embodiment, TR2, TR2-SV1 and/or TR2-SV2 polynucleotides, polypeptides, and/or agonists and/or antagonists thereofare used to treat, prevent, and/or diagnose diseases or disorders affecting or conditions associated with sinusitis.

An additional condition, disease or symptom that can be treated, prevented, and/or diagnosed by TR2, TR2-SV1 and/or TR2-SV2 polynucleotides or polypeptides, or agonists of TR2, TR2-SV1 and/or TR2-SV2, is osteomyelitis.

An additional condition, disease or symptom that can be treated, prevented, and/or diagnosed by TR2, TR2-SV1 and/or TR2-SV2 polynucleotides or polypeptides, or agonists of TR2, TR2-SV1 and/or TR2-SV2, is endocarditis.

Antagonists of TR2, TR2-SV1 and/or TR2-SV2 include binding and/or inhibitory antibodies, antisense nucleic acids, ribozymes, and TR2, TR2-SV1 and/or TR2-SV2 polypeptides of the invention. These would be expected to reverse many of the activities of the ligand described above as well as find clinical or practical application as:

A means of blocking various aspects of immune responses to foreign agents or self Examples include autoimmune disorders such as lupus, and arthritis, as well as immunoresponsiveness to skin allergies, inflammation, bowel disease, injury and pathogens. Although our current data speaks directly to the potential role of TR2 in B cell and monocyte related pathologies, it remains possible that other cell types may gain expression or responsiveness to TR2. Thus, TR2 may, like CD40 and its ligand, be regulated by the status of the immune system and the microenvironment in which the cell is located.

A therapy for preventing the B cell proliferation and immunoglobin secretion associated with autoimmune diseases such as idiopathic thrombocytopenic purpura, systemic lupus erythematosus and MS.

An inhibitor of graft versus host disease or transplant rejection.

A therapy for B cell malignancies such as ALL, Hodgkin's disease, non-Hodgkin lymphoma, Chronic lymphocyte leukemia, plasmacytomas, multiple myeloma, Burkitt's lymphoma, and EBV-transformed diseases.

A therapy for chronic hypergammaglobulinemeia evident in such diseases as monoclonalgammopathy of undetermined significance (MGUS), Waldenstrom's disease, related idiopathic monoclonalgammopathies, and plasmacytomas.

A therapy for decreasing cellular proliferation of Large B-cell Lymphomas.

A means of decreasing the involvement of B cells and immunoglobin associated with Chronic Myelogenous Leukemia.

An immunosuppressive agent(s).

TR2, TR2-SV1 and/or TR2-SV2 polypeptides or polynucleotides of the invention, or antagonists may be used to modulate IgE concentrations in vitro or in vivo.

In another embodiment, administration of TR2, TR2-SV1 and/or TR2-SV2 polypeptides or polynucleotides of the invention, or antagonists thereof, may be used to treat, prevent, and/or diagnose IgE-mediated allergic reactions including, but not limited to, asthma, rhinitis, and eczema.

An inhibitor of signaling pathways involving ERK1, COX2 and Cyclin D2 which have been associated with TR2 induced B cell activation.

The above-recited applications have uses in a wide variety of hosts. Such hosts include, but are not limited to, human, murine, rabbit, goat, guinea pig, camel, horse, mouse, rat, hamster, pig, micro-pig, chicken, goat, cow, sheep, dog, cat, non-human primate, and human. In specific embodiments, the host is a mouse, rabbit, goat, guinea pig, chicken, rat, hamster, pig, sheep, dog or cat. In preferred embodiments, the host is a mammal. In most preferred embodiments, the host is a human.

The agonists and antagonists may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as described herein.

The antagonists may be employed for instance to inhibit TR2-mediated, TR2-SV1-mediated and/or TR2-SV2-mediated chemotaxis and activation of macrophages and their precursors, and of neutrophils, basophils, B lymphocytes and some T-cell subsets, e.g., activated and CD8 cytotoxic T cells and natural killer cells, in certain auto-immune and chronic inflammatory and infective diseases. Examples of auto-immune diseases include multiple sclerosis, and insulin-dependent diabetes. The antagonists may also be employed to treat, prevent, and/or diagnose infectious diseases including silicosis, sarcoidosis, idiopathic pulmonary fibrosis by preventing the recruitment and activation of mononuclear phagocytes. They may also be employed to treat, prevent, and/or diagnose idiopathic hyper-eosinophilic syndrome by preventing eosinophil production and migration. Endotoxic shock may also be treated by the antagonists by preventing the migration of macrophages and their production of the TR2, TR2-SV1 and/or TR2-SV2 polypeptides of the present invention. The antagonists may also be employed for treating atherosclerosis, by preventing monocyte infiltration in the artery wall. The antagonists may also be employed to treat, prevent, and/or diagnose histamine-mediated allergic reactions and immunological disorders including late phase allergic reactions, chronic urticaria, and atopic dermatitis by inhibiting chemokine-induced mast cell and basophil degranulation and release of histamine. IgE-mediated allergic reactions such as allergic asthma, rhinitis, and eczema may also be treated. The antagonists may also be employed to treat, prevent, and/or diagnose chronic and acute inflammation by preventing the attraction of monocytes to a wound area. They may also be employed to regulate normal pulmonary macrophage populations, since chronic and acute inflammatory pulmonary diseases are associated with sequestration of mononuclear phagocytes in the lung. Antagonists may also be employed to treat, prevent, and/or diagnose rheumatoid arthritis by preventing the attraction of monocytes into synovial fluid in the joints of patients. Monocyte influx and activation plays a significant role in the pathogenesis of both degenerative and inflammatory arthropathies. The antagonists may be employed to interfere with the deleterious cascades attributed primarily to IL-1 and TNF, which prevents the biosynthesis of other inflammatory cytokines. In this way, the antagonists may be employed to prevent inflammation. The antagonists may also be employed to inhibit prostaglandin-independent fever induced by TR2, TR2-SV1 and/or TR2-SV2. The antagonists may also be employed to treat, prevent, and/or diagnose cases of bone marrow failure, for example, aplastic anemia and myelodysplastic syndrome. The antagonists may also be employed to treat, prevent, and/or diagnose asthma and allergy by preventing eosinophil accumulation in the lung. The antagonists may also be employed to treat, prevent, and/or diagnose subepithelial basement membrane fibrosis which is a prominent feature of the asthmatic lung. The antagonists may also be employed to treat, prevent, and/or diagnose lymphomas (e.g., one or more of the extensive, but not limiting, list of lymphomas provided herein).

TR2, TR2-SV1 and/or TR2-SV2 polynucleotides or polypeptides of the invention and/or agonists and/or antagonists thereof, may be used to treat, prevent, and/or diagnose various immune system-related disorders and/or conditions associated with these disorders, in mammals, preferably humans. Many autoimmune disorders result from inappropriate recognition of self as foreign material by immune cells. This inappropriate recognition results in an immune response leading to the destruction of the host tissue. Therefore, the administration of TR2, TR2-SV1 and/or TR2-SV2 polynucleotides or polypeptides of the invention and/or agonists and/or antagonists thereof that can inhibit an immune response, particularly the proliferation of B cells and/or the production of immunoglobulins, may be an effective therapy in treating and/or preventing autoimmune disorders. Thus, in preferred embodiments, TR2, TR2-SV1 and/or TR2-SV2 antagonists of the invention (e.g., polypeptide fragments of TR2, TR2-SV1 and/or TR2-SV2 and anti-TR2 antibodies) are used to treat, prevent, and/or diagnose an autoimmune disorder.

Autoimmune disorders and conditions associated with these disorders that may be treated, prevented, and/or diagnosed with the TR2, TR2-SV1 and/or TR2-SV2 polynucleotides, polypeptides, and/or antagonist of the invention (e.g., anti-TR2 antibodies), include, but are not limited to, autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenia purpura, autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, glomerulonephritis (e.g., IgA nephropathy), Multiple Sclerosis, Neuritis, Uveitis Ophthalmia, Polyendocrinopathies, Purpura (e.g., Henloch-Scoenlein purpura), Reiter's Disease, Stiff-Man Syndrome, Autoimmune Pulmonary Inflammation, Guillain-Barre Syndrome, insulin dependent diabetes mellitis, and autoimmune inflammatory eye disease.

Additional autoimmune disorders (that are highly probable) that may be treated, prevented, and/or diagnosed with the compositions of the invention include, but are not limited to, autoimmune thyroiditis, hypothyroidism (i.e., Hashimoto's thyroiditis) (often characterized, e.g., by cell-mediated and humoral thyroid cytotoxicity), systemic lupus erhythematosus (often characterized, e.g., by circulating and locally generated immune complexes), Goodpasture's syndrome (often characterized, e.g., by anti-basement membrane antibodies), Pemphigus (often characterized, e.g., by epidermal acantholytic antibodies), Receptor autoimmunities such as, for example, (a) Graves' Disease (often characterized, e.g., by TSH receptor antibodies), (b) Myasthenia Gravis (often characterized, e.g., by acetylcholine receptor antibodies), and (c) insulin resistance (often characterized, e.g., by insulin receptor antibodies), autoimmune hemolytic anemia (often characterized, e.g., by phagocytosis of antibody-sensitized RBCs), autoimmune thrombocytopenic purpura (often characterized, e.g., by phagocytosis of antibody-sensitized platelets.

Additional autoimmune disorders (that are probable) that may be treated, prevented, and/or diagnosed with the compositions of the invention include, but are not limited to, rheumatoid arthritis (often characterized, e.g., by immune complexes in joints), schleroderma with anti-collagen antibodies (often characterized, e.g., by nucleolar and other nuclear antibodies), mixed connective tissue disease (often characterized, e.g., by antibodies to extractable nuclear antigens (e.g., ribonucleoprotein)), polymyositis/dermatomyositis (often characterized, e.g., by nonhistone ANA), pernicious anemia (often characterized, e.g., by antiparietal cell, microsomes, and intrinsic factor antibodies), idiopathic Addison's disease (often characterized, e.g., by humoral and cell-mediated adrenal cytotoxicity, infertility (often characterized, e.g., by antispermatozoal antibodies), glomerulonephritis (often characterized, e.g., by glomerular basement membrane antibodies or immune complexes) such as primary glomerulonephritis and IgA nephropathy, bullous pemphigoid (often characterized, e.g., by IgG and complement in basement membrane), Sjogren's syndrome (often characterized, e.g. by multiple tissue antibodies, and/or a specific nonhistone ANA (SS-B)), diabetes millitus (often characterized, e.g., by cell-mediated and humoral islet cell antibodies), and adrenergic drug resistance (including adrenergic drug resistance with asthma or cystic fibrosis) (often characterized, e.g., by beta-adrenergic receptor antibodies).

Additional autoimmune disorders (that are possible) that may be treated, prevented, and/or diagnosed with the compositions of the invention include, but are not limited to, chronic active hepatitis (often characterized, e.g., by smooth muscle antibodies), primary biliary cirrhosis (often characterized, e.g., by mitchondrial antibodies), other endocrine gland failure (often characterized, e.g., by specific tissue antibodies in some cases), vitiligo (often characterized, e.g., by melanocyte antibodies), vasculitis (often characterized, e.g., by Ig and complement in vessel walls and/or low serum complement), post-MI (often characterized, e.g., by myocardial antibodies), cardiotomy syndrome (often characterized, e.g., by myocardial antibodies), urticaria (often characterized, e.g., by IgG and IgM antibodies to IgE), atopic dermatitis (often characterized, e.g., by IgG and IgM antibodies to IgE), asthma (often characterized, e.g., by IgG and IgM antibodies to IgE), inflammatory myopathies, and many other inflammatory, granulamatous, degenerative, and atrophic disorders.

In a preferred embodiment, the autoimmune diseases and disorders and/or conditions associated with the diseases and disorders recited above are treated, prevented, and/or diagnosed using anti-TR2, anti-TR2-SV1 and/or anti-TR2-SV2 antibodies.

In a specific preferred embodiment, rheumatoid arthritis is treated, prevented, and/or diagnosed using anti-TR2, anti-TR2-SV1 and/or anti-TR2-SV2 antibodies and/or other antagonist of the invention.

In a specific preferred embodiment, lupus is treated, prevented, and/or diagnosed using anti-TR2, anti-TR2-SV1 and/or anti-TR2-SV2 antibodies and/or other antagonist of the invention.

In a specific preferred embodiment, nephritis associated with lupus is treated, prevented, and/or diagnosed using anti-TR2, anti-TR2-SV1 and/or anti-TR2-SV2 antibodies and/or other antagonist of the invention.

In a specific embodiment, TR2, TR2-SV1 and/or TR2-SV2 polynucleotides or polypeptides, or antagonists thereof (e.g., anti-TR2, anti-TR2-SV1 and/or anti-TR2-SV2 antibodies) are used to treat or prevent systemic lupus erythematosus and/or diseases, disorders or conditions associated therewith. Lupus-associated diseases, disorders, or conditions that may be treated or prevented with TR2, TR2-SV1 and/or TR2-SV2 polynucleotides or polypeptides, or antagonists of the invention, include, but are not limited to, hematologic disorders (e.g., hemolytic anemia, leukopenia, lymphopenia, and thrombocytopenia), immunologic disorders (e.g., anti-DNA antibodies, and anti-Sm antibodies), rashes, photosensitivity, oral ulcers, arthritis, fever, fatigue, weight loss, serositis (e.g., pleuritus (pleuricy)), renal disorders (e.g., nephritis), neurological disorders (e.g., seizures, peripheral neuropathy, CNS related disorders), gastroinstestinal disorders, Raynaud phenomenon, and pericarditis. In a preferred embodiment, the TR2, TR2-SV1 and/or TR2-SV2 polynucleotides or polypeptides, or antagonists thereof (e.g., anti-TR2, anti-TR2-SV1 and/or anti-TR2-SV2 antibodies) are used to treat or prevent renal disorders associated with systemic lupus erythematosus. In a most preferred embodiment, TR2, TR2-SV1 and/or TR2-SV2 polynucleotides or polypeptides, or antagonists thereof (e.g., anti-TR2, anti-TR2-SV1 and/or anti-TR2-SV2 antibodies) are used to treat or prevent nephritis associated with systemic lupus erythematosus.

Similarly, allergic reactions and conditions, such as asthma (particularly allergic asthma) or other respiratory problems, may also be treated by TR2, TR2-SV1 and/or TR2-SV2 polynucleotides or polypeptides of the invention and/or agonists and/or antagonists thereof. Moreover, these molecules can be used to treat, prevent, and/or diagnose anaphylaxis, hypersensitivity to an antigenic molecule, or blood group incompatibility.

TR2, TR2-SV1 and/or TR2-SV2 polynucleotides or polypeptides of the invention and/or agonists and/or antagonists thereof, may also be used to treat, prevent, and/or diagnose organ rejection or graft-versus-host disease (GVHD) and/or conditions associated therewith. Organ rejection occurs by host immune cell destruction of the transplanted tissue through an immune response. Similarly, an immune response is also involved in GVHD, but, in this case, the foreign transplanted immune cells destroy the host tissues. The administration of TR2, TR2-SV1 and/or TR2-SV2 polynucleotides or polypeptides of the invention and/or agonists and/or antagonists thereof, that inhibits an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing organ rejection or GVHD.

Similarly, TR2, TR2-SV1 and/or TR2-SV2 polynucleotides or polypeptides of the invention and/or agonists and/or antagonists thereof, may also be used to modulate inflammation. For example, TR2, TR2-SV1 and/or TR2-SV2 polynucleotides or polypeptides of the invention and/or agonists and/or antagonists thereof, may inhibit the proliferation and differentiation of cells involved in an inflammatory response. These molecules can be used to treat, prevent, and/or diagnose inflammatory conditions, both chronic and acute conditions, including chronic prostatitis, granulomatous prostatitis and malacoplakia, inflammation associated with infection (e.g., septic shock, sepsis, or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine induced lung injury, inflammatory bowel disease, Crohn's disease, or resulting from over production of cytokines (e.g., TNF or IL-1).

In a specific embodiment, anti-TR2, anti-TR2-SV1 and/or anti-TR2-SV2 antibodies of the invention are used to treat, prevent, modulate, detect, and/or diagnose inflammation.

In a specific embodiment, anti-TR2, anti-TR2-SV1 and/or anti-TR2-SV2 antibodies of the invention are used to treat, prevent, modulate, detect, and/or diagnose inflammatory disorders.

In another specific embodiment, anti-TR2, anti-TR2-SV1 and/or anti-TR2-SV2 antibodies of the invention are used to treat, prevent, modulate, detect, and/or diagnose allergy and/or hypersensitivity.

Antibodies against TR2, TR2-SV1 and/or TR2-SV2 may be employed to bind to and inhibit TR2, TR2-SV1 and/or TR2-SV2 activity to treat, prevent, and/or diagnose ARDS, by preventing infiltration of neutrophils into the lung after injury. The agonists and antagonists of the instant may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as described hereinafter.

TR2, TR2-SV1 and/or TR2-SV2 and/or TR2 receptor polynucleotides or polypeptides of the invention and/or agonists and/or antagonists thereof, are used to treat, prevent, and/or diagnose diseases and disorders of the pulmonary system (e.g., bronchi such as, for example, sinopulmonary and bronchial infections and conditions associated with such diseases and disorders and other respiratory diseases and disorders. In specific embodiments, such diseases and disorders include, but are not limited to, bronchial adenoma, bronchial asthma, pneumonia (such as, e.g., bronchial pneumonia, bronchopneumonia, and tuberculous bronchopneumonia), chronic obstructive pulmonary disease (COPD), bronchial polyps, bronchiectasia (such as, e.g., bronchiectasia sicca, cylindrical bronchiectasis, and saccular bronchiectasis), bronchiolar adenocarcinoma, bronchiolar carcinoma, bronchiolitis (such as, e.g., exudative bronchiolitis, bronchiolitis fibrosa obliterans, and proliferative bronchiolitis), bronchiolo-alveolar carcinoma, bronchitic asthma, bronchitis (such as, e.g., asthmatic bronchitis, Castellani's bronchitis, chronic bronchitis, croupous bronchitis, fibrinous bronchitis, hemorrhagic bronchitis, infectious avian bronchitis, obliterative bronchitis, plastic bronchitis, pseudomembranous bronchitis, putrid bronchitis, and verminous bronchitis), bronchocentric granulomatosis, bronchoedema, bronchoesophageal fistula, bronchogenic carcinoma, bronchogenic cyst, broncholithiasis, bronchomalacia, bronchomycosis (such as, e.g., bronchopulmonary aspergillosis), bronchopulmonary spirochetosis, hemorrhagic bronchitis, bronchorrhea, bronchospasm, bronchostaxis, bronchostenosis, Biot's respiration, bronchial respiration, Kussmaul respiration, Kussmaul-Kien respiration, respiratory acidosis, respiratory alkalosis, respiratory distress syndrome of the newborn, respiratory insufficiency, respiratory scleroma, respiratory syncytial virus, and the like.

In a specific embodiment, TR2, TR2-SV1 and/or TR2-SV2 polynucleotides or polypeptides of the invention and/or agonists and/or antagonists thereof, are used to treat, prevent, and/or diagnose chronic obstructive pulmonary disease (COPD).

In another embodiment, TR2, TR2-SV1 and/or TR2-SV2 polynucleotides or polypeptides of the invention and/or agonists and/or antagonists thereof, are used to treat, prevent, and/or diagnose fibroses and conditions associated with fibroses, such as, for example, but not limited to, cystic fibrosis (including such fibroses as cystic fibrosis of the pancreas, Clarke-Hadfield syndrome, fibrocystic disease of the pancreas, mucoviscidosis, and viscidosis), endomyocardial fibrosis, idiopathic retroperitoneal fibrosis, leptomeningeal fibrosis, mediastinal fibrosis, nodular subepidermal fibrosis, pericentral fibrosis, perimuscular fibrosis, pipestem fibrosis, replacement fibrosis, subadventitial fibrosis, and Symmers' clay pipestem fibrosis.

The TNF family ligands are known to be among the most pleiotropic cytokines, inducing a large number of cellular responses, including cytotoxicity, anti-viral activity, immunoregulatory activities, and the transcriptional regulation of several genes (D. V. Goeddel et al., “Tumor Necrosis Factors: Gene Structure and Biological Activities,” Symp. QuanL. Biol. 51:597-609 (1986), Cold Spring Harbor; B. Beutler and A. Cerami, Annu. Rev. Biochem. 57:505-518 (1988); L. J. Old, Sci. Am. 258:59-75 (1988); W. Fiers, FEBS Lett. 285:199-224 (1991)). The TNF-family ligands, including TR2, TR2-SV1 and/or TR2-SV2 polypeptides of the present invention, induce such various cellular responses by binding to TNF-family receptors. TR2, TR2-SV1 and/or TR2-SV2 polypeptides are believed to elicit a potent cellular response including any genotypic, phenotypic, and/or morphologic change to the cell, cell line, tissue, tissue culture or patient. As indicated, such cellular responses include not only normal physiological responses to TNF-family ligands, but also diseases associated with increased apoptosis or the inhibition of apoptosis. Apoptosis-programmed cell death-is a physiological mechanism involved in the deletion of peripheral B and/or T lymphocytes of the immune system, and its disregulation can lead to a number of different pathogenic processes (J. C. Ameisen, AIDS 8:1197-1213 (1994); P. H. Krammer et al., Curr. Opin. Immunol. 6:279-289 (1994)).

Diseases associated with increased cell survival, or the inhibition of apoptosis that may be diagnosed, treated, or prevented with the TR2, TR2-SV1 and/or TR2-SV2 polynucleotides or polypeptides of the invention, and agonists and antagonists thereof, include cancers (such as follicular lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors, including, but not limited to, colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer); autoimmune disorders (such as systemic lupus erythematosus and immune-related glomerulonephritis rheumatoid arthritis); viral infections (such as herpes viruses, pox viruses and adenoviruses); inflammation; graft vs. host disease; acute graft rejection and chronic graft rejection. Thus, in preferred embodiments TR2, TR2-SV1 and/or TR2-SV2 polynucleotides or polypeptides of the invention and/or agonists or antagonists thereof, are used to treat, prevent, and/or diagnose autoimmune diseases and/or inhibit the growth, progression, and/or metastasis of cancers, including, but not limited to, those cancers disclosed herein, such as, for example, lymphocytic leukemias (including, for example, MLL and chronic lymphocytic leukemia (CLL)) and follicular lymphomas. In another embodiment TR2, TR2-SV1 and/or TR2-SV2 polynucleotides or polypeptides of the invention are used to activate, differentiate or proliferate cancerous cells or tissue (e.g., B cell lineage related cancers (e.g., CLL and MLL), lymphocytic leukemia, or lymphoma) and thereby render the cells more vulnerable to cancer therapy (e.g., chemotherapy or radiation therapy).

Moreover, in other embodiments, TR2, TR2-SV1 and/or TR2-SV2 polynucleotides or polypeptides of the invention or agonists or antagonists thereof, are used to inhibit the growth, progression, and/or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyo sarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.

Diseases associated with increased apoptosis apoptosis that may be diagnosed, treated, or prevented with the TR2, TR2-SV1 and/or TR2-SV2 polynucleotides or polypeptides of the invention, and agonists and antagonists thereof, include AIDS; neurodegenerative disorders (such as Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration); myelodysplastic syndromes (such as aplastic anemia), ischemic injury (such as that caused by myocardial infarction, stroke and reperfusion injury), toxin-induced liver disease (such as that caused by alcohol), septic shock, cachexia and anorexia. Thus, in preferred embodiments TR2, TR2-SV1 and/or TR2-SV2 polynucleotides or polypeptides of the invention and/or agonists or antagonists thereof, are used to treat, prevent, and/or diagnose the diseases and disorders listed above.

In preferred embodiments, TR2, TR2-SV1 and/or TR2-SV2 polypeptides of the invention and/or agonists or antagonists thereof (e.g., anti-TR2 antibodies) inhibit the growth of human histiocytic lymphoma U-937 cells in a dose-dependent manner. In additional preferred embodiments, TR2, TR2-SV1 and/or TR2-SV2 polypeptides of the invention and/or agonists or antagonists thereof (e.g., anti-TR2 antibodies) inhibit the growth of PC-3 cells, HT-29 cells, HeLa cells, MCF-7 cells, and A293 cells. In highly preferred embodiments, TR2, TR2-SV1 and/or TR2-SV2 polynucleotides or polypeptides of the invention and/or agonists or antagonists thereof (e.g., anti-TR2 antibodies) are used to inhibit growth, progression, and/or metastasis of prostate cancer, colon cancer, cervical carcinoma, and breast carcinoma.

Thus, in additional preferred embodiments, the present invention is directed to a method for enhancing apoptosis induced by a TNF-family ligand, which involves administering to a cell which expresses a TR2, TR2-SV1 and/or TR2-SV2 receptor an effective amount of TR2, TR2-SV1 and/or TR2-SV2, or an agonist or antagonist thereof, capable of increasing or decreasing TR2, TR2-SV1 and/or TR2-SV2 mediated signaling. Preferably, TR2, TR2-SV1 and/or TR2-SV2 mediated signaling is increased or decreased to treat, prevent, and/or diagnose a disease wherein decreased apoptosis or decreased cytokine and adhesion molecule expression is exhibited. An agonist or antagonist can include soluble forms of TR2, TR2-SV1 and/or TR2-SV2 and monoclonal antibodies directed against the TR2, TR2-SV1 and/or TR2-SV2 polypeptide.

In a further aspect, the present invention is directed to a method for inhibiting apoptosis induced by a TNF-family ligand, which involves administering to a cell which expresses the TR2, TR2-SV1 and/or TR2-SV2 receptor an effective amount of an agonist or antagonist capable of increasing or decreasing TR2, TR2-SV1 and/or TR2-SV2 mediated signaling. Preferably, TR2, TR2-SV1 and/or TR2-SV2 mediated signaling is increased or decreased to treat, prevent, and/or diagnose a disease wherein increased apoptosis or NF-kappaB expression is exhibited. An agonist or antagonist can include soluble forms of TR2, TR2-SV1 and/or TR2-SV2 and monoclonal antibodies directed against the TR2, TR2-SV1 and/or TR2-SV2 polypeptide.

Because TR2, TR2-SV1 and TR2-SV2 belong to the TNF superfamily, the polypeptides should also modulate angiogenesis. In addition, since TR2, TR2-SV1 and TR2-SV2 inhibit immune cell functions, the polypeptides will have a wide range of anti-inflammatory activities. TR2, TR2-SV1 and/or TR2-SV2 may be employed as an anti-neovascularizing agent to treat, prevent, and/or diagnose solid tumors by stimulating the invasion and activation of host defense cells, e.g., cytotoxic T cells and macrophages and by inhibiting the angiogenesis of tumors. Those of skill in the art will recognize other non-cancer indications where blood vessel proliferation is not wanted. They may also be employed to enhance host defenses against resistant chronic and acute infections, for example, mycobacterial infections via the attraction and activation of microbicidal leukocytes. TR2, TR2-SV1 and/or TR2-SV2 may also be employed to inhibit T-cell proliferation by the inhibition of IL-2 biosynthesis for the treatment of T-cell mediated auto-immune diseases and lymphocytic leukemias (including, for example, chronic lymphocytic leukemia (CLL)). TR2, TR2-SV1 and/or TR2-SV2 may also be employed to stimulate wound healing, both via the recruitment of debris clearing and connective tissue promoting inflammatory cells. In this same manner, TR2, TR2-SV1 and/or TR2-SV2 may also be employed to treat, prevent, and/or diagnose other fibrotic disorders, including liver cirrhosis, osteoarthritis and pulmonary fibrosis. TR2, TR2-SV1 and/or TR2-SV2 also increases the presence of eosinophils that have the distinctive function of killing the larvae of parasites that invade tissues, as in schistosomiasis, trichinosis and ascariasis. It may also be employed to regulate hematopoiesis, by regulating the activation and differentiation of various hematopoietic progenitor cells, for example, to release mature leukocytes from the bone marrow following chemotherapy, i.e., in stem cell mobilization. TR2, TR2-SV1 and/or TR2-SV2 may also be employed to treat, prevent, and/or diagnose sepsis.

Polynucleotides and/or polypeptides of the invention and/or agonists and/or antagonists thereof are useful in promoting angiogenesis, wound healing (e.g., wounds, burns, and bone fractures) and regulating hematopoiesis. Polynucleotides and/or polypeptides of the invention and/or agonists and/or antagonists thereof are also useful as an adjuvant to enhance immune responsiveness to specific antigen, anti-viral immune responses.

More generally, polynucleotides and/or polypeptides of the invention and/or agonists and/or antagonists thereof are useful in regulating (i.e., elevating or reducing) immune response. For example, polynucleotides and/or polypeptides of the invention may be useful in preparation or recovery from surgery, trauma, radiation therapy, chemotherapy, and transplantation, or may be used to boost immune response and/or recovery in the elderly and immunocompromised individuals. Alternatively, polynucleotides and/or polypeptides of the invention and/or agonists and/or antagonists thereof are useful as immunosuppressive agents, for example in the treatment or prevention of autoimmune disorders. In specific embodiments, polynucleotides and/or polypeptides of the invention are used to treat or prevent chronic inflammatory, allergic or autoimmune conditions, such as those described herein or are otherwise known in the art.

Preferably, treatment using TR2, TR2-SV1 and/or TR2-SV2 polynucleotides or polypeptides, and/or agonists or antagonists of TR2, TR2-SV1 and/or TR2-SV2 (e.g., anti-TR2 antibody), could either be by administering an effective amount of TR2, TR2-SV1 and/or TR2-SV2 polypeptide of the invention, or agonist or antagonist thereof, to the patient, or by removing cells from the patient, supplying the cells with TR2, TR2-SV1 and/or TR2-SV2 polynucleotide, and returning the engineered cells to the patient (ex vivo therapy). Moreover, as further discussed herein, the TR2, TR2-SV1 and/or TR2-SV2 polypeptide or polynucleotide can be used as an adjuvant in a vaccine to raise an immune response against infectious disease.

The agonists and antagonists may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as described herein.

All of the above described applications may be used in veterinary medicine, as well as in human treatment regimens.

The above-recited applications have uses in a wide variety of hosts. Such hosts include, but are not limited to, human, murine, rabbit, goat, guinea pig, camel, horse, mouse, rat, hamster, pig, micro-pig, chicken, goat, cow, sheep, dog, cat, non-human primate, and human. In specific embodiments, the host is a mouse, rabbit, goat, guinea pig, chicken, rat, hamster, pig, sheep, dog or cat. In preferred embodiments, the host is a mammal. In most preferred embodiments, the host is a human.

Cardiovascular Disorders

TR2 polynucleotides, polypeptides, agonists or antagonists of the invention may be used to treat cardiovascular disorders, including peripheral artery disease, such as limb ischemia.

Cardiovascular disorders include cardiovascular abnormalities, such as arterio-arterial fistula, arteriovenous fistula, cerebral arteriovenous malformations, congenital heart defects, pulmonary atresia, and Scimitar Syndrome. Congenital heart defects include aortic coarctation, cortriatriatum, coronaryvessel anomalies, crisscross heart, dextrocardia, patent ductus arteriosus, Ebstein's anomaly, Eisenmenger complex, hypoplastic left heart syndrome, levocardia, tetralogy of fallot, transposition ofgreat vessels, double outlet right ventricle, tricuspid atresia, persistent truncus arteriosus, and heart septal defects, such as aortopulmonary septal defect, endocardial cushion defects, Lutembacher's Syndrome, trilogy of Fallot, ventricular heart septal defects.

Cardiovascular disorders also include heart disease, such as arrhythmias, carcinoid heart disease, high cardiac output, low cardiac output, cardiac tamponade, endocarditis (including bacterial), heart aneurysm, cardiac arrest, congestive heart failure, congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, heart hypertrophy, congestive cardiomyopathy, left ventricular hypertrophy, right ventricular hypertrophy, post-infarction heart rupture, ventricular septal rupture, heart valve diseases, myocardial diseases, myocardial ischemia, pericardial effusion, pericarditis (including constrictive and tuberculous), pneumopericardium, postpericardiotomy syndrome, pulmonary heart disease, rheumatic heart disease, ventricular dysfunction, hyperemia, cardiovascular pregnancy complications, Scimitar Syndrome, cardiovascular syphilis, and cardiovascular tuberculosis.

Arrhythmias include sinus arrhythmia, atrial fibrillation, atrial flutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branch block, sinoatrial block, long QT syndrome, parasystole, Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome, Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, and ventricular fibrillation. Tachycardias include paroxysmal tachycardia, supraventricular tachycardia, accelerated idioventricular rhythm, atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia, ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia, sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.

Heart valve disease include aortic valve insufficiency, aortic valve stenosis, hear murmurs, aortic valve prolapse, mitral valve prolapse, tricuspid valve prolapse, mitral valve insufficiency, mitral valve stenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonary valve stenosis, tricuspid atresia, tricuspid valve insufficiency, and tricuspid valve stenosis.

Myocardial diseases include alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvular stenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardial fibrosis, Kearns Syndrome, myocardial reperfusion injury, and myocarditis.

Myocardial ischemias include coronary disease, such as angina pectoris, coronary aneurysm, coronary arteriosclerosis, coronary thrombosis, coronary vasospasm, myocardial infarction and myocardial stunning.

Cardiovascular diseases also include vascular diseases such as aneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis, Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome, Sturge-Weber Syndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis, aortitis, Leriche's Syndrome, arterial occlusive diseases, arteritis, enarteritis, polyarteritis nodosa, cerebrovascular disorders, diabetic angiopathies, diabetic retinopathy, embolisms, thrombosis, erythromelalgia, hemorrhoids, hepatic veno-occlusive disease, hypertension, hypotension, ischemia, peripheral vascular diseases, phlebitis, pulmonary veno-occlusive disease, Raynaud's disease, CREST syndrome, retinal vein